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J. Biol. Chem., Vol. 282, Issue 45, 32877-32889, November 9, 2007

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Functional Interaction of Neuronal Ca_v1.3 L-type Calcium Channel with Ryanodine Receptor Type 2 in the Rat Hippocampus^*

Sunoh Kim, Hyung-Mun Yun, Ja-Hyun Baik, Kwang Chul Chung^¶, Seung-Yeol Nah^||, and Hyewhon Rhim¹

From the Life Sciences Division, Korea Institute of Science and Technology, Seoul 136-791, Graduate School of Biotechnology, Korea University, Seoul 136-701, the ^¶Department of Biology, College of Science, Yonsei University, Seoul 120-749, and the ^||Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea

Received for publication, February 16, 2007 , and in revised form, August 29, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Neuronal L-type Ca²⁺ channels do not support synaptic transmission, but they play an essential role in synaptic activity-dependent gene expression. Ca_v1.2 and Ca_v1.3 are the two most widely expressed L-type Ca²⁺ channels in neurons and have different biophysical and subcellular distributions. The function of the Ca_v 1.3 L-type Ca²⁺ channel and its cellular mechanisms in the central nervous system are poorly understood. In this study, using a yeast two-hybrid assay, we found that the N terminus of the rat Ca_v1.3 ₁ subunit interacts with a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2). Reverse transcription-PCR and Western blot assays revealed high expression of both Ca_v1.3 and RyR2 in the rat hippocampus. We also demonstrate a physical association of Ca_v1.3 with RyR2 using co-immunoprecipitation assays. Moreover, immunocytochemistry revealed prominent co-localization between Ca_v1.3 and RyR2 in hippocampal neurons. Depolarizing cells by an acute treatment of a high concentration of KCl (high-K, 60 mM) showed that the activation of L-type Ca²⁺ channels induced RyR opening and led to RyR-dependent Ca²⁺ release, even in the absence of extracellular Ca²⁺. Furthermore, we found that RyR2 mRNA itself is increased by long term treatment of high-K via activation of L-type Ca²⁺ channels. These acute and long term effects of high-K on RyRs were selectively blocked by small interfering RNA-mediated silencing of Ca_v1.3. These results suggest a physical and functional interaction between Ca_v1.3 and RyR2 and important implications of Ca_v1.3/RyR2 clusters in translating synaptic activity into alterations in gene expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Intracellular Ca²⁺ plays an important role in Ca²⁺-regulated neuronal functions, including membrane excitability, neurotransmitter release, and synaptic plasticity (1, 2). It is not only controlled by influx of Ca²⁺ into the cell through voltage-dependent Ca²⁺ channels (VDCCs)² but also by the release of Ca²⁺ from intracellular stores. VDCCs can be distinguished by their electrophysiological and pharmacological characteristics as follows: high voltage-activated channels that are further divided into L-, N-, P/Q-, and R-type Ca²⁺ channels and low voltage-activated (T-type) Ca²⁺ channels (3, 4). Among the high voltage-activated Ca²⁺ channels, physiological functions of N- and P/Q-type Ca²⁺ channels in the central nervous system (CNS) have been extensively studied because of their central role in controlling Ca²⁺ entry into, and thus neurotransmitter release from, neuronal synaptic terminals (5, 6). Neuronal L-type Ca²⁺ channels have also been studied particularly because of their importance in translating synaptic activity into alterations in gene expression and neuronal function (7–10). Until recently, four genes, which encode L-type Ca²⁺ channel pore-forming subunits, had been identified. These were designated Ca_v1.1 (_1S), Ca_v1.2 (_1C), Ca_v1.3 (_1D), and- Ca_v1.4 (_1F) (11, 12). Among them, Ca_v1.2 and Ca_v1.3 are the two most widely expressed L-type Ca²⁺ channel subunits in neurons and have different biophysical properties and subcellular distributions (12–14). However, most studies of L-type Ca²⁺ channels are confined to Ca_v1.2, and surprisingly little is known about the neuronal function of Ca_v1.3 in the CNS. Until recently, this was mainly because of a lack of selective pharmacological tools, selective agonist or antagonist, for distinguishing Ca_v1.2 and Ca_v1.3 subtypes.

To elucidate the neuronal function of Ca_v1.3 and its cellular mechanisms in the CNS, we employed a yeast two-hybrid screen of the rat brain cDNA library with the N terminus of rat Ca_v1.3 ₁ subunit (Ca_v1.3-NT) as bait and isolated a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2-NT) as a binding partner. Ryanodine receptors (RyRs) are a multigene family of channel proteins that mediate Ca²⁺ release from intracellular Ca²⁺ stores such as the endoplasmic reticulum. Three different genes coding for isoforms of RyRs have been identified and cloned (RyR1, RyR2, and RyR3). For all three isoforms of RyRs, mRNA has been detected in the brain, with RyR2 mRNA showing the most abundant expression (15, 16). Interestingly, it has been reported that spatial learning induces changes in RyR2 mRNA and protein in the rat hippocampus (17, 18). In muscle, the function of RyRs is extremely important for triggering muscle contraction. It is now widely accepted that RyRs are physically linked with L-type Ca²⁺ channels, providing an amplification of the Ca²⁺ signal necessary to trigger contraction (19, 20). However, this coupling between L-type Ca²⁺ channels and RyRs has not been well characterized in the CNS.

In this study, we demonstrate a physical interaction between the Ca_v1.3 ₁ subunit and RyR2 using yeast two-hybrid, co-immunoprecipitation, and immunocytochemistry assays. In addition, we demonstrate that the activation of L-type Ca²⁺ channels, especially via Ca_v1.3, is also functionally coupled with an increase in intracellular Ca²⁺ from RyR-sensitive Ca²⁺ stores, and a further increase in the RyR mRNA level in rat hippocampal neurons.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmid Construction and Yeast Two-hybrid Methods—Full-length rat neuronal Ca_v 1.3 cDNA, GenBank^TM accession number AF370009 [GenBank] (21), was kindly provided by Dr. D. Lipscombe (Brown University, Providence, RI). A yeast two-hybrid screen was performed using Matchmaker GAL4 two-hybrid system 3 (Clontech). To construct bait, cDNA fragments encoding the N terminus of Ca_v1.3 (Ca_v1.3-NT) cDNA (amino acids 1–151) were subcloned into the NdeI/SalI sites of Gal4 DNA-binding domain vector, pGBKT7, and then bait plasmid was transformed into yeast strain AH109. The rat brain cDNA library in GAL4 activation domain vector pACT2 (prey) was kindly provided by Dr. P. Voisin (University of Poitiers, France) and further transformed into yeast strain Y187. For the yeast two-hybrid screen of rat brain cDNA library with Ca_v1.3-NT cDNA, the corresponding AH109 yeast strains (MAT) were mated with the Y187 yeast strain (MATa). The diploid colonies were plated on a nutritionally selective plate deficient in adenine, histidine, leucine, and tryptophan (-Ade, -His, -Leu, -Trp) to screen the library. False positives were eliminated using two reporters, ADE2 and HIS3, and MEL1 encoding -galactosidase was assayed on X--gal indicator plates. Doubly positive clones were isolated and characterized by DNA sequencing. -Galactosidase activities for a yeast two-hybrid assay were measured using a colony lift assay in accordance with the manufacturer's instructions (Clontech). Briefly, yeast cells were lifted onto a Whatman No. 5 filter paper, permeabilized by immersion in liquid nitrogen for 20 s, thawed at room temperature, and placed onto two sheets of pre-wetted filter papers in Z-buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, and 50 mM -mercaptoethanol) containing 5-bromo-4 chloro-3-indolyl-D-galatopyranoside (1 mg/ml).

Hippocampal Cell Cultures—Cultured hippocampal neurons were prepared using the technique modified from Kim et al. (22). Briefly, the hippocampi were isolated from 16–18-day-old fetal Sprague-Dawley rats and incubated with 0.25% trypsin in Leibovitz L-15 medium (Invitrogen) for 20 min at 37 °C. Cells were then mechanically dissociated with fire-polished Pasteur pipettes by trituration and plated on poly-L-lysine-coated coverslips or culture dishes. Cultures were maintained in Neurobasal/B27 medium (Invitrogen) containing 0.5 mM L-glutamine, 25 µM 2-mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin under a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Because astrocyte-conditioned medium (ACM) is known to facilitate neuronal differentiation and network formation in hippocampal neurons (23), we added ACM into Neurobasal/B27 medium through an entire culture procedure. ACM was generated by the incubation of a confluent carpet of astrocyte type 1 according to the method reported previously (24), stored at -70 °C, and added to Neurobasal/B27 medium before use. Experiments were carried out on neurons after 5–10 days in vitro (DIV).

Cell Transfection—Hippocampal neurons were transfected after 5 DIV using the Lipofectamine 2000 (Invitrogen) according to the modified manufacturer's instructions. Briefly, the Ca_v1.3-NT construct in pCMV-2B vector (Clontech) or each siRNA and the Lipofectamine 2000 diluted in B27-free Neurobasal medium were combined and incubated at room temperature for 20 min. Culture media were removed and saved; the DNA or siRNA/Lipofectamine 2000 mixture was added to the cells, and the dishes were incubated at 37 °C for 2 h. The saved culture media were then returned to the dish, and the culture was returned to the incubator for 48 h. To monitor transfection efficiency, the GFP expression vector pEGFP-N1 (Clontech) was co-transfected with the Ca_v1.3-NT construct or each siRNA. For each Ca²⁺ imaging experiment, the transfected cells without GFP vector were loaded with Fura-2/AM after separately confirming transfection efficiency (>80%).

Intracellular Ca²⁺ Imaging—The acetoxymethyl ester form of fura-2 (fura-2/AM, Molecular Probes, Eugene, OR) was used as the fluorescent Ca²⁺ indicator. Cells were incubated for 40–60 min at room temperature with 5 µM fura-2/AM and 0.001% Pluronic F-127 in a HEPES-buffered solution composed of 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES, and 10 mM glucose, pH adjusted to 7.4 with NaOH. The cells were then washed with HEPES-buffered solution and placed on an inverted microscope (Olympus, Japan). The cells were illuminated using a xenon arc lamp, and the required excitation wavelengths (340 and 380 nm) were selected by means of a computer-controlled filter wheel (Sutter Instruments, Novato, CA). Emitter fluorescence light was reflected through a 515-nm long pass filter to a frame transfer cooled CCD camera, and the ratios of emitted fluorescence were calculated using a digital fluorescence analyzer. All imaging data were collected and analyzed using Meta Imaging software (Molecular Devices, Downingtown, PA). Saline with 60 mM KCl (high-K) was made by replacing an equivalent amount of NaCl.

Neuronal Cell Death Assay—Hippocampal cell cultures were washed with a HEPES-buffered solution, and then incubated with 60 mM KCl for 30 min to 2 h at 37 °C. Neuronal cell viability was then measured by the detection of dehydrogenase activity that was retained in living cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (25). An aliquot (50 µl) of MTT solution (1 mg/ml) in PBS was directly added to the cultures, and the cultures were then incubated for 4 h to allow MTT to metabolize to formazan. The supernatant was then aspirated, and 100 µl of Me₂SO was added to dissolve the formazan. Absorbances were measured with an automated spectrophotometric plate reader at a wavelength of 560 nm. Cell viability was expressed as relative percentages in comparison with untreated controls.

Total RNA Isolation and RT-PCR Analysis—The total RNA from each sample was extracted using easy-BLUE^TM kit (iNtRON Biotech, Korea) according to the manufacturer's instructions. Total RNA samples were treated with DNase I before cDNA synthesis and quantified using ultraviolet spectrophotometry. Single strand cDNAs were then synthesized in a reverse transcription reaction in the presence of 2 µg of total RNA, 0.1 nmol of specific antisense primer, and the first-strand cDNA synthesis mix (iNtRON Biotech) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 10 mM dithiothreitol, 0.25 mM of each dNTP, and 100 units of Moloney murine leukemia virus reverse transcriptase. The sequences of the sense (+) and antisense (-) primer pairs used for specific amplification of rat L-type Ca²⁺ channels, RyRs, and controls were as follows: Ca_v1.2 (+)5'-GACAACCTGGCTGATGCGGAGAGCCTGAC-3' and (-)5'-ATGCGGTGGCACTGCAGGCGGAACCTG-3' (GenBank^TM accession number NM_012517 [GenBank] , product size 401 bp); Ca_v1.3 (+)5'-GGTCACTTTGCTCCGACGTATCCAGCC-3' and (-)5'-GTTTGGAGTCTTCTGGTTCGTCATCTT-3' (GenBank^TM accession number NM_017298 [GenBank] , product size 500 bp); RyR1 (+)5'-CGGGCCGTGAACTTCTGCT-3' and (-)5'-CTGTCAGGAATGGAACCACT-3' (GenBank^TM accession number AF112256 [GenBank] , product size 150 bp); RyR2 (+)5'-CTACTCAGGATGAGGTGCGA-3' and (-)5'-CTCTCTTCAGATCCAAGCCA-3' (GenBank^TM accession number AF112257 [GenBank] , product size 157 bp); RyR3 (+) 5'-GTGTCAAACTAGTCATTGCCAA-3' and (-)5'-ATCCTGTCATCTGTAACTCACAA-3' (GenBank^TM accession number XM_342491 [GenBank] , product size 445 bp); phosphoglycerate kinase 1 (PGK1) (+)5'-AGGTGCTCAACAACATGGAG-3' and (-) 5'-TACCAGAGGCCACAGTAGCT-3' (conserved sequence of human, rat, and mouse, product size 183 bp); and -actin (+)5'-GTCACCAACTGGGACGACATG-3' and (-) 5'-GCCGTCAGGCAGCTCGTAGC-3' (conserved sequence of human, rat, and mouse, product size 510 bp). Using these primers, amplification of cDNA fragments was performed on PCR through 25–28 cycles with 1 µl of each RT product as a template DNA in 60 mM Tris-HCl (pH 9.1) buffer containing 18 mM (NH₄)₂SO₄, 16 mM MgCl₂, 0.25 mM of each dNTP, 0.1 nmol of each primer, and Ex-TaqDNA polymerase (Takara, Japan). Each sample (5 µl) of final PCR product was separated using a 1.5–2.5% agarose gel and visualized using UV fluorescence after staining with ethidium bromide for 15 min. Template controls, -actin or PGK1, were included in each run.

Small Interfering RNA—siRNA sequences of rat Ca_v1.2 (sense, 5'-CGUCCUUGCUGAACUCACUdTdT-3', and antisense, 3'-dTdTGCAGGAACGACUUGAGUGA-5'; NM_012517 [GenBank] ), Ca_v1.3 (sense, 5'-CGAGAGAGGUCAAAGGUGAdTdT-3', and antisense, 3'-dTdTGCUCUCUCCAGUUUCCACU-5'; NM_017298 [GenBank] ), and scrambled siRNA (negative, sense, 5'-CCUACGCCACCAAUUUCGUdTdT-3', and antisense, 3'-dTdTGGAUGCGGUGGUUAAAGCA-5') were designed using siRNA target finder software (Turbo si-Designer, Bioneer, Korea), and two single-strand RNA oligonucleotides were chemically synthesized and annealed to form the siRNA duplex. Each siRNA at the concentration of 5 pmol/µl was transferred into primary cultures of rat hippocampal neurons (5 DIV) using Lipofectamine 2000 (Invitrogen).

Co-immunoprecipitation and Western Blot Analysis—Age- and weight-controlled adult male Sprague-Dawley rats (60–70 days old, 230–260 g) were used for co-immunoprecipitation and Western blot analysis. After the cerebral cortex, thalamus, cerebellum, hippocampus, and spinal cord were isolated, they were separately frozen in liquid nitrogen, ground, and homogenized in 1 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM MgCl₂, 0.5% Triton X-100, and complete protease inhibitor mixture). The solution was incubated on ice for 1 h and centrifuged for 15 min (4 °C) at 15,000 x g, and the supernatant was collected. The concentration of total protein was determined by the modified Bradford method (Bio-Rad). Co-immunoprecipitation assays were performed using the ProFound mammalian co-immunoprecipitation kit (Pierce). Briefly, lysates were precleared by incubation with the control gel component for 4 h at 4 °C. The precleared lysates were then added to antibody-coupled gels containing anti-Ca_v1.2 (Alomone Labs, Jerusalem, Israel), anti-Ca_v1.3 (Alomone Labs), anti-RyR2 (clone c3-33, Sigma), or rabbit IgG (Jackson ImmunoResearch, West Grove, PA) antibody. After incubation for 16 h at 4 °C, immunoprecipitates were washed four times with the immunoprecipitation buffer and once with reduced salt immunoprecipitation buffer (125 mM NaCl) before elution. For the detection of RyR2, protein samples were separated on a 3–12% gradient SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was first blocked with PBS containing 5% skim milk and 0.1% Tween 20 for 1 h. After three washes, the membranes were incubated with anti-Ca_v1.2 (1:150 dilution), anti-Ca_v1.3 (1:150 dilution), or anti-RyR2 (1:100 dilution) antibodies for 20 h at room temperature in PBS containing 1% skim milk and 0.1% Tween 20. For a positive control, anti--actin antibodies (1:2500 dilution, Sigma) were incubated for 1 h. After three washes, the membranes were incubated with peroxidase-conjugated secondary antibodies in PBS containing 3% skim milk and 0.1% Tween 20 for 2 h at room temperature. The signal resulting from the immunoreactivity was detected using Western blot detection reagent (Elipis Biotech, Korea).

Immunocytochemistry—For immunocytochemistry, cultured hippocampal neurons were grown on culture slides (BD Biosciences), fixed in 4% paraformaldehyde and PBS for 20 min at 4 °C, and washed three times in PBS. The cells were permeabilized with 0.2% Triton X-100 in PBS for 30 min, washed three times in PBS, and then blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. The cells were then incubated for 12 h at 4 °C with anti-Ca_v1.3 (1:200) and anti-RyR2 (1:100) antibodies. Next, the cells were washed three times and incubated with FITC- and rhodamine-conjugated secondary antibodies (1:200; Abcam, Cambridge, UK) for 2 h at room temperature. The cells were washed three times, mounted on glass slides using CRYSTAL/MOUNT^TM (Biomeda Corp., Foster City, CA), and viewed on a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan).

Expression of Recombinant Proteins and Pulldown Assay—GST-NT, GST-NT1, GST-NT2, and GST-NT3 plasmids were constructed by cloning Ca_v1.3-NT (amino acids: 1–151), NT1 (amino acids: 1–60), NT2 (amino acids: 45–115), and NT3 (amino acids: 90–151) domains from pGBKT7/NT into the BamHI/EcoRI sites of the pGEX4T-1 vector (Amersham Biosciences). GST, GST-NT, GST-NT1, GST-NT2, and GST-NT3 plasmids were transformed into the bacterial host strain BL21 (DE3) and induced by adding 0.5 mM isopropyl 1-thio--D-galactopyranoside at 37 °C during midlog phase. The cells were sonicated in lysis buffer (1x PBS (pH 7.4), 1 mM dithiothreitol, 0.01% Triton X-100, and protease inhibitor mixture). GST, GST-NT, GST-NT1, GST-NT2, and GST-NT3 proteins were immobilized by glutathione-agarose 4B. For GST pulldown assays using rat hippocampal lysates, hippocampal lysates were prepared from age- and weight-controlled adult male Sprague-Dawley rats (60–70 days old and 230–260 g). Rat hippocampi were homogenized in 1 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM MgCl₂, 0.5% Triton X-100, and complete protease inhibitor mixture). The solution was incubated on ice for 1 h and centrifuged for 15 min (4 °C) at 15,000 x g, and the supernatant was collected. GST pulldown assays were performed using the Profound pulldown GST protein-protein interaction kit (Pierce). Immobilized GST, GST-NT, GST-NT1, GST-NT2, and GST-NT3 proteins were incubated with prepared hippocampal lysates. Bound proteins were eluted by boiling for 10 min at 95 °C in SDS sample buffer followed by immunoblotting with anti-RyR2 (1:100 dilution) and anti-GST antibodies (1:5000 dilution; Novagen).

Statistical Analysis—To quantify the results obtained from RT-PCR analysis, the optic density of RT-PCR product bands was measured using the AphaEase program (version 5.1, Alpha Innotech, San Leandro, CA) and analyzed using GraphPad Prism version 4 program (GraphPad Software, Inc., San Diego, CA). Statistical significance was performed on the data using unpaired Student's t test or one-way analysis of variance. A p value of <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of RyR2 as a Ca_v1.3 Binding Protein Using the Yeast Two-hybrid System—To identify the function of Ca_v1.3 L-type Ca²⁺ channels and its cellular mechanism in the CNS, we performed a yeast two-hybrid screen using Ca_v1.3-NT as bait (Fig. 1A). After screening the rat brain cDNA library, we identified that RyR2-NT interacted with Ca_v1.3-NT. To confirm this specific interaction, we carried out a yeast two-hybrid assay using Ca_v1.3-NT cDNA (bait) transformed in the AH109 yeast strain and RyR2-NT cDNA (prey) transformed into the Y187 yeast strain. When both the AH109 and Y187 yeast strains were mated, a blue color was detected with a -galactosidase colony-lift filter assay (Fig. 1B), suggesting a positive interaction between Ca_v1.3-NT-(1–151) and RyR2-NT-(3150–3680).

Expression Profiles of Ca_v1.3 and RyRs in the Rat CNS—Before characterizing the physical and functional interaction between Ca_v1.3 and RyR2, we examined the expression profiles of Ca_v1.2, Ca_v1.3, and RyRs (RyR1, RyR2, and RyR3) in the adult rat brain to find brain regions in which both proteins are highly expressed. Using RT-PCR analysis, we observed that RyR2 mRNA is the most abundant of the three RyRs isoforms in the CNS (Fig. 2A). In particular, the hippocampus had a significantly high level of RyR2 mRNA expression. Although it is not as prominent as RyR2, the high level of Ca_v1.3 mRNA was also detected in the thalamus, cerebellum, and hippocampus, whereas the expression level of Ca_v1.2 mRNA was high in the cortex and thalamus. The expression profiles for Ca_v1.2, Ca_v1.3, and RyR2 proteins were also examined using a Western blot assay (Fig. 2B). When anti-Ca_v1.3 and anti-RyR2 antibodies were used to recognize the Ca_v1.3 ₁ subunit (260 kDa) and RyR2 (565 kDa), relatively high expression was detected in the hippocampus. However, the expression level of Ca_v1.2 was very low in the hippocampus as compared with Ca_v1.3 and RyR2 when the Ca_v1.2 ₁ subunit (240 kDa) was detected using anti-Ca_v1.2 antibodies. These results demonstrate high expression of both proteins in the hippocampus, and thus, we chose this structure to further study the physical and functional interaction between Ca_v1.3 and RyR2.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Interaction between Cav1.3-NT and RyR2-NT in a yeast two-hybrid assay. A, schematic diagram of neuronal Cav1.3 1 subunit indicating the intracellular domains (NT (N terminus), I-II loop, II-III loop, III-IV loop, and CT (C terminus)). Amino acid boundaries of the intracellular domains are indicated in parentheses. B, results from -galactosidase colony-lift filter assay. Cav1.3-NT (bait, 1–151, left) and RyR2-NT (prey, 3150–3680, center) constructs were transformed into the yeast strain AH109 and Y187 and selected on Trp- and Leu-deficient media, respectively. The mated yeast colony was spotted and grown on nutritionally selective plates (-Ade, -Leu, -His, and -Trp, right) and analyzed using -galactosidase activities.

View larger version (80K): [in this window] [in a new window]   FIGURE 2. Expression profiles of Cav1.3 and RyRs in the rat CNS. A, expression levels of Cav1.2, Cav1.3, and RyRs (RyR1, RyR2, and RyR3) mRNAs were determined using RT-PCR analysis in the five regions (Cx, cerebral cortex; Th, thalamus; Cb, cerebellum; Hp, hippocampus; Sp, spinal cord) of the rat CNS. The bar graph indicates the mean values of relative mRNA levels from each group (n = 5) when the level of each mRNA in the cerebral cortex was counted as an arbitrary control (100%), and a background optical density was used as a negative control (0%). **, p < 0.01 compared with RyR2 mRNA level in cerebral cortex. B, expression levels of Cav1.2 (240 kDa), Cav1.3 (260 kDa), and RyR2 (565 kDa) proteins were determined by anti-Cav1.2, anti-Cav1.3, and anti-RyR2 antibodies using Western blot analysis, respectively. -Actin was used as a positive control for RT-PCR and Western blot analyses. C, developmental expression profiles of Cav1.3, RyR1, RyR2, and RyR3 mRNA using RT-PCR from embryonic day 18 (E18) to postnatal (P) day 70 (adult, Ad), counting the day after overnight mating as E0 and the day of birth as P1. PGK1 was used as a positive amplification control for RT-PCR analysis. The experiments shown in Fig. 2 were repeated in more than triplicate with similar results.

RyR2 Interacts with the N Terminus of Ca_v1.3 in Vitro—Next, we attempted to determine whether Ca_v1.3-NT selectively binds to RyR2 in rat hippocampal neurons using a GST pulldown assay. After GST and Ca_v1.3-NT fused with GST (GST-Ca_v1.3-NT) were expressed in Escherichia coli, GST and GST-Ca_v1.3-NT were immobilized by glutathione-agarose 4B. Adult rat hippocampal lysates were incubated with the glutathione-agarose 4B bound to GST or GST-Ca_v1.3-NT. As shown in Fig. 4B, GST-Ca_v1.3-NT was interacted with endogenous hippocampal RyR2. When used a negative control, GST alone was determined not to interact with RyR2. We next determined to identify the specific residues within Ca_v1.3-NT for binding with RyRs. After Ca_v1.3-NT was cut into three constructs (NT1, 1–60; NT2, 45–115; NT3, 90–151, see Fig. 4A), GST pulldown assays were again performed with three GST fused constructs, GST-NT1, GST-NT2, and GST-NT3. As shown in Fig. 4, C–E, we found that RyR2 interacted with GST-NT2, but not GST-NT1 nor GST-NT3. These data indicate that the NT2 domain, 45–115-amino acid region of Ca_v1.3-NT, contains the binding sites for RyR2 in vitro.

Physiological Interaction between L-type Ca²⁺ Channels and RyRs in Cultured Rat Hippocampal Neurons—In muscle, L-type Ca²⁺ channels act as voltage sensors to control ryanodine-sensitive Ca²⁺ stores in the sarcoplasmic reticulum by excitation-contraction coupling (26), and it is known that these effects occur partly via extracellular Ca²⁺-independent pathways (27). Therefore, we examined if L-type Ca²⁺ channels and RyRs physiologically interact in cultured rat hippocampal neurons as has been shown in muscle cells. First, we examined whether VDCCs, RyRs, and inositol 1,4,5-trisphosphate (InsP₃) receptors functionally operate in our culture system using fura-2-based digital imaging techniques. In most cultured hippocampal neurons, the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by the acute application (20 s) of a high concentration of KCl (60 mM, high-K) because of membrane depolarization, by ryanodine receptor activation by caffeine, or by Ca²⁺ release by ATP in an InsP₃-dependent manner in a normal HEPES-buffered solution containing 2.5 mM Ca²⁺ (Fig. 5A). Ryanodine is used as a ryanodine receptor agonist/antagonist depending on the applied concentration, as it acts as an antagonist at high concentrations (>10 µM). When 20 µM ryanodine was pretreated for 1 min, ryanodine inhibited the caffeine-induced [Ca²⁺]_i increase (87.3 ± 2.5% inhibition, n = 10) with no effect on ATP-induced [Ca²⁺]_i increase (0.9 ± 1.4% inhibition, n = 10). These results suggest that VDCCs, RyRs, and InsP₃ receptors are present and functionally active in our culture system.

View larger version (52K): [in this window] [in a new window]   FIGURE 3. Cav1. 3 interacts with RyR2 in vivo and co-localizes with RyR2 in rat hippocampal neurons. A–D, in vivo co-immunoprecipitation (IP) assays in the adult rat hippocampus. Where specified, solubilized total proteins (100 µg) from the hippocampus were immunoprecipitated with anti-Cav1.3, anti-Cav1.2, or anti-RyR2 antibodies (Ab). For RyR2 proteins, the immune complexes were then analyzed by 3–12% gradient SDS-PAGE, followed by immunoblotting with anti-RyR2 antibodies (A and B). For Cav1.3 or Cav1.2 proteins, the immune complexes were resolved by 6% SDS-PAGE and analyzed by immunoblotting with anti-Cav1.3 (C) or anti-Cav1.2 antibodies (D). The proper expression of each endogenous protein in cell lysates (Lysate) was identified by immunoblotting with anti-Cav1.2, anti-Cav1.3, or anti-RyR2 antibodies as indicated. N1, N2, and M indicate the negative control 1 using nonimmobilized control gel, the negative control 2 using IgG·Bind gel, and protein markers, respectively. E, confocal microscope-based immunofluorescence analysis showing co-localization of Cav1.3 and RyR2 proteins in cultured rat hippocampal neurons. Cav1.3 (green, left) was immunostained with rabbit anti-Cav1.3 and then FITC-conjugated secondary antibodies. RyR2 (red, middle) was immunostained with mouse anti-RyR2 and then rhodamine-conjugated secondary antibodies. Right panels show the merged images of the left and middle panels. Negative control experiments (Con) were processed with only FITC- or rhodamine-conjugated IgG antibodies in cultured hippocampal neurons. The experiments shown in Fig. 3 were repeated in triplicate with similar results.

To confirm a specific physical interaction between Ca_v1.3 and RyRs in high-K/Bay K-mediated RyR-sensitive Ca²⁺ release, we examined how Ca²⁺ influx in response to high-K/Bay K is modulated by overexpression of Ca_v1.3-NT domain (residues 1–151) in cultured hippocampal neurons. Compared with control cells, Ca_v1.3-NT-overexpressed cells produced a significant decrease in high-K/Bay K-mediated Ca²⁺ increase in Ca²⁺-free solution (73.4 ± 2.2% inhibition, n = 13; Fig. 5E). These results suggest that the physical interaction between Ca_v1.3-NT and RyRs is essential to mediate RyR-sensitive Ca²⁺ release upon high-K/Bay K-mediated L-type Ca²⁺ channel activations.

View larger version (82K): [in this window] [in a new window]   FIGURE 4. Cav1.3-NT interacts with RyR2 in rat hippocampus in vitro. A, schematic diagram of Cav1.3-NT and three overlapped fragments of Cav1.3-NT (NT1, 1–60; NT2, 45–115; and NT3, 90–151). Cav1.3-NT, NT1, NT2, and NT3 are schematically depicted with their respective sequence coordinates. B–E, selective binding between Cav1.3-NT and RyR2 using a GST pulldown assay. After GST, GST-Cav1.3-NT, GST-NT1, GST-NT2, and GST-NT3 fusion proteins were expressed in E. coli, they were analyzed using 12% SDS-PAGE and immunoblotting with an anti-GST antibody. Proteins in hippocampal lysates (Hp) were allowed to bind to GST-Cav1.3-NT (B), GST-NT1 (C), GST-NT2 (D), or GST-NT3 (E) fusion proteins. The elution fractions were resolved by a 3–12% gradient SDS-PAGE followed by immunoblotting with an anti-RyR2 antibody. For each experiment, GST alone was used a negative control. The experiments shown here were repeated in triplicate with similar results.

L-type Ca²⁺ Channel-mediated Long Term Effects on RyR2 mRNA in Cultured Hippocampal Neurons—The most important role of L-type Ca²⁺ channels in the CNS is to alter gene expression by modulating synaptic activity (7–10). Recent data indicate that both neuronal L-type Ca²⁺ channels and RyRs play critical roles in hippocampus-dependent synaptic plasticity and spatial memory (28–30). The involvement of L-type Ca²⁺ channels on synaptic plasticity could be due to modulated gene expression, which might be necessary for synaptic plasticity and memory formation such as cAMP-response element-binding protein (CREB) (31–36). To expand our understanding of the cellular mechanisms of L-type Ca²⁺ channels in synaptic plasticity, we examined whether long term activation of L-type Ca²⁺ channels directly modulates RyR2 gene expression in addition to its acute effect on RyR-sensitive Ca²⁺ release.

We initially set up the experimental conditions for long term activation of L-type Ca²⁺ channels on RyR2 expression using fura-2-based digital imaging techniques. When cells were treated with high-K in a normal HEPES-buffered solution, [Ca²⁺]_i was transiently increased to produce a peak (2.6 ± 0.1 of 340/360 nm ratio) but declined to reach a sustained plateau over 15 min, and the plateau was maintained for 2 h of treatment (1.4 ± 0.1 of 340/360 nm ratio, n = 24; data not shown). When the cells were treated with 10 µM nifedipine, nifedipine significantly inhibited both the peak and sustained plateau of high-K-induced [Ca²⁺]_i increase by 49.6 ± 3.5 and 70.5 ± 2.3%, respectively (n = 44; Fig. 7, A and B), suggesting that most of the sustained [Ca²⁺]_i increases were mediated by an activation of L-type Ca²⁺ channels. The involvement of L-type Ca²⁺ channels was further confirmed by the treatment of Bay K; 10 µM Bay K increased the sustained level of [Ca²⁺]_i by 24.4 ± 5.0% (n = 18). Interestingly, the sustained plateau of high-K-induced [Ca²⁺]_i increase was inhibited by 20 µM ryanodine (30.1 ± 3.6% inhibition, n = 17), suggesting that L-type Ca²⁺ channels and RyR-sensitive Ca²⁺ release mostly contributed to a sustained [Ca²⁺]_i increase produced by the long term treatment of high-K in cultured hippocampal neurons.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. L-type Ca2+ channel-mediated [Ca2+]i increase by acute treatment of high-K in cultured rat hippocampal neurons. A, in most cultured hippocampal cells, the acute application (20 s) of 60 mM KCl (high-K), 5 mM caffeine (Caff), or 100 µM ATP produced a rapid increase of [Ca2+]i in a normal HEPES-buffered solution recorded using fura-2-based digital imaging techniques. Caffeine-induced [Ca2+]i increase was decreased by a 1-min pretreatment of 20 µM ryanodine (Ry) as the antagonist of RyRs, whereas the ATP-induced signal was not changed by 20 µM ryanodine. B, application of high-K also produced an [Ca2+]i increase in 0 Ca2+, 2 mM EGTA containing HEPES-buffered (0-Ca2+ or extra Ca2+-free) solution. This [Ca2+]i increase was inhibited by 20 µM ryanodine and potentiated by the selective L-type Ca2+ channel agonist 10 µM Bay K-8644 (Bay K), which was also inhibited by 20 µM ryanodine. C, pooled results illustrating the change of 340/380 nm ratio by acute treatment with high-K and various drugs (n = 11). ***, p < 0.001. D, effect of the intracellular calcium chelator BAPTA-AM. The high-K/Bay K-induced [Ca2+]i increase in Ca2+-free solution was completely inhibited by a 30-min loading of 10 µM BAPTA-AM (n = 9). E, overexpression of Cav1.3-NT domain (1–151) in cultured hippocampal neurons. Compared with empty vector (pCMV-2B), transfected control cells (vector), Cav1.3-NT-overexpressed cells produced a significant decrease in high-K/Bay K-mediated Ca2+ increase in Ca2+-free solution (73.4 ± 2.2% inhibition, n = 13).

Specific Involvement of Ca_v1.3 in the Expression of RyR2 mRNA—The significant increase in RyR2 mRNA by high-K was again confirmed at 2 h (133.5 ± 3.2%, n = 6, ^**, p < 0.01), and it was significantly reduced by co-treatment with 10 µM nifedipine (78.7 ± 1.1%, n = 6, ^***, p < 0.001; Fig. 8A), suggesting that most of the increase occurred through activation of L-type Ca²⁺ channels. Curiously, the addition of nifedipine to high-K produced RyR2 levels that were below base line. Given that nifedipine has multiple targets (37, 38), we examined the effect of nifedipine itself and found no effect on RyR2 mRNA levels by nifedipine itself (10 µM; Fig. 7F). The level of RyR2 mRNA was also increased by 10 µM Bay K (132.9 ± 3.2%, n = 6, ^**, p < 0.01) by itself, but not further by high-K with Bay K (133.1 ± 1.4%, n = 6, ^**, p < 0.01), suggesting that the level of RyR2 mRNA is fully activated by either high-K-induced membrane depolarization or by the L-type Ca²⁺ channel activator Bay K. This increase of RyR2 mRNA was modulated by a RyR agonist (5 mM caffeine) or antagonist (20 µM ryanodine); 20 µM ryanodine decreased high-K-, high-K/Bay K-, or caffeine-induced increases in RyR2 mRNA. These results suggested the involvement of L-type Ca²⁺ channels and RyR-sensitive Ca²⁺ stores in high-K-induced RyR2 mRNA increase.

View larger version (43K): [in this window] [in a new window]   FIGURE 6. Specific physiological interaction between Cav1.3 and RyRs. A and B, to knock down L-type Ca2+ channels selectively, primary hippocampal cultures were exposed to siRNA against 1 subunits of Cav1.2 (A, -Cav1.2) and Cav1.3 (B, -Cav1.3). As a negative control siRNA (Neg), a nontargeting scrambled siRNA with no sequence homology to any known gene sequences was used. The expression of Cav1.2 or Cav1.3 siRNA was validated using RT-PCR at 48 h after the transfection of siRNA (n = 3). C, in control cells, the acute application (20 s) of high-K/Bay K (10 µM) for the full activation of L-type Ca2+ channels produced a rapid increase of [Ca2+]i in a normal HEPES-buffered solution. This [Ca2+]i increase was inhibited by 10 µM nifedipine (Nif, 69.9 ± 5.0%, n = 4). The application of high-K/Bay K also produced a [Ca2+]i increase in a Ca2+-free solution, which was inhibited by 20 µM ryanodine. D and E, in Cav1.2 knockdown cells (D) and Cav1.3 knockdown cells (E), high-K/Bay K-induced [Ca2+]i increase was inhibited by nifedipine, although the degree was decreased by selective knockdown of the Cav1.2 or Cav1.3 gene. In Cav1.2 knockdown cells, high-K/Bay K still produced an [Ca2+]i increase in Ca2+-free solution, which was inhibited by ryanodine (n = 7). However, in Cav1.3 knockdown cells, high-K/Bay K-induced [Ca2+]i increase was very small in Ca2+-free solution (n = 6). F, in Cav1.2/1.3 double knockdown cells, there is no effect by nifedipine because of total knockdown of L-type Ca2+ channels, and Ca2+ increase by high-K/Bay K was hardly detected in extra Ca2+-free solution (n = 6).

It has been reported recently that activation of NMDA receptors mediates CREB phosphorylation and gene expression via L-type Ca²⁺ channels in rat striatal neurons (44, 45). Here we examined whether membrane depolarization via activation of NMDA receptors mediates RyR2 mRNA expression. Omitting Mg²⁺ in a HEPES-buffered solution induced an increase of RyR2 mRNA (142.8 ± 1.9%, n = 6, ^**, p < 0.01) in cultured hippocampal neurons (Fig. 8C) because it has been known that an exposure of neuronal cultures to Mg²⁺-free bath solution induces NMDA receptor-dependent oscillations of membrane potential and [Ca²⁺]_i (46, 47). The treatment of 50 µM NMDA for 20 min significantly increased RyR2 mRNA in the absence of Mg²⁺ (154.7 ± 2.5%, n = 6, ^**, p < 0.01) in cultured hippocampal neurons. The time of NMDA treatment was decreased to 20 min because we previously observed NMDA-mediated cell death during longer time periods (22). This elevation of RyR2 mRNA was also significantly blocked by nifedipine (10 µM, 104.3 ± 1.8%, n = 6, ^***, p < 0.001) and ryanodine (20 µM, 107.4 ± 1.9%, n = 6, ^***, p < 0.001). The competitive (APV) and noncompetitive (MK-801) antagonists of the NMDA receptor significantly blocked the NMDA-induced increase of RyR2 mRNA. The mean values of relative mRNA levels were 104.7 ± 0.8% (n = 6) and 94.7 ± 1.0% (n = 6) by 100 µM APV and 1 µM MK-801, respectively. Taken together, these results suggest that membrane depolarization via high-K or NMDA could induce RyR2 gene expression via the L-type Ca²⁺ channel- and RyR-sensitive pathways in cultured hippocampal neurons.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Increase of RyR2 expression via long term treatment of high-K. A, changes of [Ca2+]i levels by long term treatment of hippocampal neurons with high-K stimulation along with 10 µM Bay K or 10 µM nifedipine (Nif). B, bar graph shows pooled results illustrating the change of 340/380 ratios at sustained plateaus by long term treatment (2 h) with high-K and various drugs (n = 17–44). C, time-dependent increase of RyR2 mRNA in rat primary hippocampal cultures using RT-PCR analysis. The level of RyR2 mRNA was increased at 15 min after the treatment of high-K and remained after 2 h of treatment in a normal HEPES-buffered solution. The values from the bar graph were expressed as percentages (mean ± S.E.) when calculated relative to each control (0 min, open bar, 100%). D, increase of RyR2 protein in rat primary hippocampal cultures using Western blot analysis. The level of RyR2 protein was increased at 12 h after 2 h of treatment of high-K in a normal HEPES-buffered solution. The values from the bar graph were expressed as percentages (mean ± S.E.) when calculated relative to each control (open bar, 100%). E, effect of high-K on cell viability after treatment for the indicated time. The cell viability accessed using the MTT assay showed no significant effect for up to 2 h of treatment with high-K. F, effects of 1 µM ionomycin (Iono) and 10 µM nifedipine (Nif) on RyR2 mRNA using RT-PCR analysis. Cells were treated with drugs for 2 h. **, p < 0.01 and NS (not significant) when compared with control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we first identified a direct physical interaction between Ca_v1.3 with RyR2 in the hippocampus, and we described the evidence for functional modulation of the L-type Ca²⁺ channel on RyR-sensitive Ca²⁺ release and RyR2 gene expression. Three different RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues (48), and all three have also been known to be present in neurons (15, 49, 50). RyR1, the skeletal-muscle subtype, is poorly represented in the brain but is present in discrete isolated neurons of the cerebellum, cortex, and hippocampus. RyR2, the cardiac subtype, shows the most abundant expression as the major brain isoform. RyR3 is also ubiquitously expressed in the brain but in lower amounts. The level of [Ca²⁺]_i can be transiently increased by two mechanisms as follows: Ca²⁺ influx through Ca²⁺-permeable channels in the plasma membrane or the mobilization of Ca²⁺ from intracellular Ca²⁺ stores. In muscle, both paths are coupled, providing an amplification of the Ca²⁺ signal necessary to trigger contraction. However, skeletal muscle and cardiac muscle both differ in the subtypes of channels involved and in the mechanism of coupling. It is now widely accepted that Ca_v1.1 and RyR1 in skeletal muscle and Ca_v1.2 and RyR2 in cardiac muscle are physically linked and functionally coupled to trigger contraction (19, 20).

In the CNS, this specific coupling between L-type Ca²⁺ channels and RyRs has had limited reporting in a few studies (51–53). Chavis et al. (51) first reported the functional coupling between RyRs and L-type Ca²⁺ channels in neurons, but they did not specify the subtypes of channels involved. Using co-immunoprecipitation, so far only two cases reported this physical link as a protein complex as follows: the interaction between Ca_v1.2 and RyR1 in whole rat brain (52) and the specific interaction between RyR1/Ca_v1.2 and RyR2/Ca_v1.3 in rat spinal cord (53). Although Mouton et al. (52) mentioned the possible interaction between Ca_v1.3 and RyR1 in rat spinal cord, they did not provide clear evidence to support this specific interaction in the brain. Furthermore, when Ouardouz et al. (53) reported their results, they mentioned that it was quite an unexpected finding when compared with what was known in muscle. However, our results strongly support this previous study and demonstrate the specific interaction between RyR2 with Ca_v1.3 in the rat hippocampus using co-immunoprecipitation. This specific interaction in the hippocampus is significant because it is not only the center of spatial memory-related behavior and electrophysiological studies such as long term potentiation and long term depression, but it is also a region of the brain where molecular mechanisms of synaptic plasticity related with gene expression have been extensively studied.

View larger version (46K): [in this window] [in a new window]   FIGURE 8. Increase of RyR2 mRNA via an activation of Cav 1.3 L-type Ca2+ channels. A, cultures were exposed to high-K, nifedipine (10 µM), Bay K (10 µM), ryanodine (20 µM), or caffeine (Caff, 5 mM) in a normal HEPES-based solution (Extra-Ca2+) and harvested at 2 h after the addition of each drug. B, cultures were exposed to high-K, nifedipine (Nif), Bay K, and ryanodine (Ry) in extra Ca2+-free solution and harvested at 2 h after the addition of each drug. C, primary hippocampal cultures were switched to Mg2+-free external solution and exposed to NMDA (50 µM), nifedipine (10 µM), ryanodine (20 µM), APV (100 µM), or MK-801 (1 µM) and harvested at 20 min after the addition of each drug. D, specific role of Cav1.3 in high-K-induced RyR2 gene expression in cultured hippocampal neurons. The levels of RyR2 mRNA in Cav1.2, Cav1.3, or Cav1.2/1.3 knockdown cells are shown. Neg, scrambled sequence of negative-siRNA. Cells were treated with high-K for 2 h. The values from the bar graph were expressed as percentages (mean ± S.E.) when calculated relative to each control (Con, open bar, 100%). ***, p < 0.001; **, p < 0.01 when compared with control.

The third and last piece of evidence is functional modulation of L-type Ca²⁺ channels on RyRs. After we found that acute activation of L-type Ca²⁺ channels opened RyRs and increased [Ca²⁺]_i from RyR-sensitive stores, which supports the physiological interaction between them, we demonstrated that long term activation of L-type Ca²⁺ channels by high-K or NMDA treatment increased RyR2 mRNA. The novelty of our data lies in isolating a specific role of Ca_v1.3 between two types of neuronal L-type Ca²⁺ channels, Ca_v1.2 and Ca_v1.3, by utilizing siRNA technology. Based on these experiments, we found that acute and long term effects of high-K were mostly inhibited by Ca_v1.3 knockdown and Ca_v1.2/Ca_v1.3 double knockdown, but not in control, negative siRNA-transfected and Ca_v1.2 knockdown cells. However, based on the observations showing a lesser degree of high-K/Bay K-mediated Ca²⁺ increase in Ca_v1.2 siRNA-treated cells than in controls and residual Ca²⁺ increase in Ca_v1.3 siRNA-treated cells (Fig. 6, D and E), we cannot completely rule out any contribution of Ca_v1.2 to the RyR coupling.

How is the acute effect of high-K relevant to the long term effects on RyR2 mRNA? There are many Ca²⁺-dependent genes that are regulated in expression by the increase of [Ca²⁺]_i. It is possible that increases in cytoplasmic Ca²⁺ levels increase RyR2 mRNA. However, we suggest that this is not the case, and it occurs by the activation of both L-type Ca²⁺ channels, especially Ca_v1.3, and RyRs under the following circumstances. First, the use of a Ca²⁺ ionophore such as ionomycin did not increase RyR mRNA, suggesting that a simple increase of cytoplasmic Ca²⁺ levels cannot account for the increase in RyR2 mRNA. Second, both membrane depolarization and activation of RyR-sensitive Ca²⁺ release are required for an increase in RyR2 mRNA, suggesting high-K-induced membrane depolarization activates L-type Ca²⁺ channels and triggers ryanodine-sensitive Ca²⁺ release. NMDA-induced membrane depolarization also increased RyR2 mRNA, but its signal also required the involvement of both L-type Ca²⁺ channels and RyRs. Third, the specific blockade both on acute and long term effects using Ca_v1.3, but not Ca_v1.2, knockdown cells, supports that the high-K-induced increase in RyR2 mRNA occurs in Ca_V1.3-specific pathways and not by a simple increase in cytoplasmic Ca²⁺ levels.

How could an activation of L-type Ca²⁺ channels trigger RyR-sensitive Ca²⁺ release in the absence of extracellular Ca²⁺? One possible mechanism is that L-type Ca²⁺ channels act as a voltage sensor to activate RyRs or induce a direct activation of RyRs by conformational coupling with L-type Ca²⁺ channels. As it is now widely accepted that Ca_v1.1 and RyR1 in skeletal muscle and Ca_v1.2 and RyR2 in cardiac muscle are physically linked and functionally coupled to trigger contraction (19, 20), it is highly possible that Ca_v1.3 and RyR2 are also physically linked and functionally coupled in hippocampus neurons. Therefore, acute and long term treatments of high-K increase [Ca²⁺]_i by both activation of Ca_v1.3 and RyR2 could further modulate gene expression, including RyR2 itself in this study. However, more experiments have to be performed to examine the exact role of RyR2 in membrane depolarization-induced [Ca²⁺]_i increase, particularly when selective antagonists of each RyRs become available.

What is the physiological significance of the interaction between Ca_v1.3 and RyR2 in the hippocampus? The spatiotemporal change of [Ca²⁺]_i levels is critically involved in higher brain activities, including learning and memory. Many studies indicate that neuronal L-type Ca²⁺ channels are particularly important in translating synaptic activity into alterations in gene expression and neuronal function (7–10). Recent data indicate that Ca_v1.2 serves a critical function in hippocampus-dependent spatial memory by coupling NMDA receptor-independent synaptic activity to transcriptional events (28). In addition, biochemical processes, such as the modulation of CREB, extracellular signal-regulated protein kinase (ERK), and nuclear factor of activated T-cells (NF-AT), have been known to link to Ca_v1.2 L-type Ca²⁺ channel activity (31–34). However, very little is known about genes modulated by Ca_v1.3 L-type Ca²⁺ channels in the CNS. On the other hand, Ca²⁺ release from intracellular stores via RyRs is also reported to be required for the induction of long term potentiation and long term depression in the hippocampus (29, 30). The specific role of RyR2 among RyRs was reported by Zhao et al. (18); spatial learning induced changes in RyR2 mRNA and protein levels in the rat hippocampus. Therefore, our results provide a possible role of Ca_v1.3 L-type Ca²⁺ channels in hippocampal synaptic plasticity via a novel linkage with RyR2. However, further investigation is necessary to understand the exact role of Ca_v1.3 in regulating the RyR2 gene using Ca_v1.3 ₁ subunit knock-out (Ca_v1.3₁ ^-/-) mice.

In conclusion, using a yeast two-hybrid screening and functional studies, we obtained results supporting that Ca_v1.3 and RyR2 are present as physically associated forms, and the activation of Ca_v1.3 induces [Ca²⁺]_i increase from RyR-sensitive Ca²⁺ stores and increases RyR2 mRNA levels. Similar to excitation-contraction coupling in skeletal muscle, these results suggest that a functional coupling of Ca_v1.3 and RyR2 may play a vital role in an amplification of the Ca²⁺ signal into neuronal function, including regulating synaptic efficacy and gene expression.

	FOOTNOTES

 
^* This work was supported by KIST Core-Competence Program and Brain Research Center of the 21st Century Frontier Research Program Grant M103KV010007-07K2201-00710 (to H. R.), the 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.

¹ To whom correspondence should be addressed: Life Sciences Division, Korea Institute of Science and Technology, 39-1 Hawholgok-dong, Sungbuk-gu, Seoul 136-791, Korea. Tel.: 82-2-958-5923; Fax: 82-2-958-5909; E-mail: hrhim{at}kist.re.kr.

² The abbreviations used are: VDCC, voltage-dependent Ca²⁺ channel; CNS, central nervous system; RyR2, ryanodine receptor type 2; siRNA, small interfering RNA; RyR, ryanodine receptor; Ca_v1.3-NT, N terminus of Ca_v1.3 ₁ subunit; RyR2-NT, a partial N-terminal amino acid sequence of RyR2; [Ca²⁺]_i, intracellular Ca²⁺ concentration; InsP₃, inositol 1,4,5-triphophate; Bay K, Bay K-8644; NMDA, N-methyl-D-aspartate; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; DIV, days in vitro; high-K, high concentration of KCl; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT, reverse transcription.

	ACKNOWLEDGMENTS

 
We thank Dr. D. Lipscombe and Dr. P. Voisin for providing rat neuronal Ca_v1.3 cDNA and rat brain cDNA library, respectively.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION