Direct Interaction and Functional Coupling between Metabotropic Glutamate Receptor Subtype 1 and Voltage-sensitive Cav2.1 Ca2+ Channel*
Jun Kitano
,
Motohiro Nishida
,
Yuko Itsukaichi
¶,
Itsunari Minami ||,
Masaaki Ogawa
,
Tomoo Hirano ||,
Yasuo Mori
¶ and
Shigetada Nakanishi
**
From the
Department of Biological Sciences, Faculty of Medicine, and Department of Molecular and System Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, the
Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, and ¶School of Life Science, Graduate University for Advanced Studies, Okazaki 444-0864, and the ||Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
Received for publication, March 29, 2003
, and in revised form, April 16, 2003.
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ABSTRACT
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Intracellular Ca2+ concentrations ([Ca2+]i) are regulated in a spatiotemporal manner via both entry of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Metabotropic glutamate receptor subtype 1 (mGluR1) is a G protein-coupled receptor that stimulates the inositol 1,4,5-trisphosphate-Ca2+ signaling cascade, whereas Cav2.1 is a pore-forming channel protein of P/Q-type voltage-sensitive Ca2+ channels. In this investigation, we showed that mGluR1 and Cav2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. This assembly occurs through the direct interaction between their carboxyl-terminal intracellular domains. Calcium imaging and whole-cell recording showed that mGluR1 inhibits Cav2.1-mediated [Ca2+]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary
2/
and
1 subunits, respectively. This inhibition occurred in a ligand-independent manner and was enhanced by pre-activation of mGluR1 in a ligand-dependent manner. In contrast, simultaneous stimulation of mGluR1 and Cav2.1 induced large [Ca2+]i increases. Furthermore, the temporally regulated inhibition and stimulation of [Ca2+]i increases by mGluR1 and Cav2.1 were observed at dendrites but not soma of cultured Purkinje neurons. These data suggest that the assembly of mGluR1 and Cav2.1 provides the mechanism that ensures spatiotemporal regulation of [Ca2+]i in glutamatergic neurotransmission.
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INTRODUCTION
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Excitatory neurotransmitter glutamate triggers an increase in intracellular calcium concentration ([Ca2+]i) both through extracellular Ca2+ influx and intracellular Ca2+ mobilization (13). Glutamate interacts with two distinct types of receptors, namely, ionotropic and metabotropic glutamate receptors (4). The
-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type ionotropic receptors depolarizes membrane potentials and induces Ca2+ influx through voltage-sensitive Ca2+ channels (VSCCs)1 (3, 5). Glutamate also activates N-methyl-D-aspartic acid-type ionotropic receptors and enhances Ca2+ influx through these calcium-permeable receptors (14). Metabotropic glutamate receptors (mGluRs) are the family of G protein-coupled receptors (4, 6). Eight different subtypes of mGluRs exist, and these are classified into three groups (4, 6). Group 1 mGluRs (mGluR1 and mGluR5) stimulate phosphatidylinositol hydrolysis to generate the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (7, 8). IP3 mobilizes intracellular Ca2+ through interaction with IP3 receptors (9). The glutamate-mediated increase in [Ca2+]i plays an important role in regulating a variety of neuronal processes, including neural development, synaptic plasticity, and neuronal cell degeneration (1012).
Several Ca2+ signaling molecules are located in close proximity and linked through protein-protein interaction. This assembly of Ca2+ signaling molecules contributes to efficient activation of specific Ca2+ signaling cascades and cross-talk between different Ca2+ signaling pathways (3, 1214). Cav2.1 is a pore forming membrane protein of VSCC and mediates P-type and P/Q-type Ca2+ currents with auxiliary
/
and
subunits (1518). Both Cav2.1 and mGluR1a, a large splice variant of mGluR1, are highly expressed in the Purkinje neurons (PNs) in the cerebellum (1924). Gene targeting showed that both Cav2.1 and mGluR1 are important for the network formation and function of the cerebellum (18, 25, 26). Furthermore, coincident stimulation of mGluR1 and VSCC has been shown to potentiate [Ca2+]i increase in PNs (27). The coincident rise in [Ca2+]i is important for integrating synaptic inputs to PNs (2, 28, 29). In contrast, the stimulation of group 1 mGluRs has been shown to inhibit P/Q-type Ca2+ channels in cortical and globus pallidus neurons (30, 31). However, neither temporally regulated mechanisms of mGluR1-mediated P/Q-type Ca2+ channel modulation nor the physical interaction between mGluR1 and P/Q-type Ca2+ channels have been addressed yet. In this investigation, we report that mGluR1a and Cav2.1 form a protein complex through direct interaction between these two proteins. We also show that the heteromeric assembly of mGluR1a and Cav2.1 plays an important role in temporally regulated [Ca2+]i increase in both cultured PNs and heterologously expressing cells.
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EXPERIMENTAL PROCEDURES
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ImmunohistochemistryAll procedures were performed according to guidelines of Kyoto University Faculty of Medicine. Immunohistochemistry of mouse cerebellar sections (40 µm) was performed with use of rabbit anti-mGluR1a antibody (32) and rabbit anti-Cav2.1 antibody (Alomone and Sigma) as described previously (33, 34). PNs were cultured as described previously (35). For immunostaining of cultured PNs, the cells were first reacted with the human antibody against the extracellular region of mGluR1 (36) and fixed with 1% paraformaldehyde for 3 min. The fixed cells were permeabilized, blocked, and reacted with rabbit anti-Cav2.1 antibody. Immunoreactive signals were visualized with Alexa568-conjugated anti-rabbit IgG (Molecular Probes) and fluorescein isothiocyanate-conjugated anti-human IgG (Caltag). For double-immunofluorescence staining, cerebella were cut into sections of 30-µm thickness before fixation. The sections were fixed with 4% paraformaldehyde and 1% picric acid in 0.1 M phosphate buffer (pH 7.4) for 10 min and reacted with human anti-mGluR1 antibody (36) and rabbit anti-Cav2.1 antibody. Immunoreactive signals were visualized with Alexa488-conjugated anti-human IgG and Alexa594-conjugated anti-rabbit IgG antibodies (Molecular Probes).
Immunoprecipitation, Immunoblotting, and Pull-down AssayFor COS-7 cell expression, Myc/His tag was introduced into the carboxyl-terminal end of rabbit Cav2.1 (BI-2 clone, GenBankTM CAA40715
[GenBank]
) (16) by PCR. Myc/His-tagged Cav2.1 (Cav2.1-Myc-His) was inserted into the pcDNA3.1 mammalian expression vector (Invitrogen). The mammalian expression vector for rat mGluR1a was described previously (37). Expressed mGluR1a and Cav2.1-Myc-His were immunoprecipitated with rabbit anti-mGluR1a (32) and anti-Myc (Santa Cruz Biotechnology) antibodies, respectively. The immunoprecipitates were immunoblotted with mouse anti-His (Qiagen) and anti-mGluR1a (BD Transduction) antibodies. For in vitro pull-down assays, the whole carboxyl-terminal region of rat mGluR1a (amino acid residues 8411199, GenBankTM CAA40799
[GenBank]
) was fused to the carboxyl-terminal end of His/FLAG-tagged enhanced green fluorescent protein (EGFP, Clontech) (HFG-1CT), and HFG-1CT was inserted into pFASTBAC (Invitrogen). Other intracellular regions of rat mGluR1a and rabbit Cav2.1 were constructed by PCR and inserted in-frame into pET-32a (Novagen) and pGEX-4T-1 (Amersham Biosciences), respectively; the regions used for rat mGluR1a were as follows: 1CT-1, residues 841932; 1CT-2, 9331013; 1CT-3, 10351073; and 1CT-4, 10801199; the regions used for rabbit Cav2.1 were as follows: CavNT, residues 198; CavIII, 361487; CavIIIII-1, 7151036; CavIIIII-2, 10111253; CavIIIIV, 15211575; CavCT, 18212424; CavCTa, 19752424; CavCTb, 18212024; and CavCTc, 18212164. Proper in-frame insertions and the absence of any sequence errors of all PCR products were confirmed by DNA sequencing. The HFG-1CT protein was produced with use of the Bac-to-Bac baculovirus expression system (Invitrogen) and HighFive cells and purified with nickel-nitrilotriacetic acid-agarose (Qiagen). GST fusion proteins were expressed in Escherichia coli and purified with glutathione-Sepharose 4B beads. The concentrations of purified proteins were determined by DC protein assay kit (Bio-Rad). His-tagged thioredoxin fusion proteins were expressed in E. coli and purified with nickel-nitrilotriacetic acid-agarose. The purified proteins we used were more than 90% of purity as analyzed by Coomassie Brilliant Blue R250 staining after PAGE. One microgram of the purified proteins was used for both protein immobilization and protein binding in pull-down assays. The procedures for transfection, protein purification, immunoprecipitation, immunoblotting, and GST pull-down assays were described previously (38).
Calcium Imaging and Electrophysiological Recording of HEK293 CellsHEK293 cells expressing the BI-2 form of Cav2.1 together with the auxiliary
2/
and
1 subunits (HEK-Cav) were established as described previously (39), except that the pKCRH2-neo vector (40) was used for expression of BI cDNA. The mammalian expression vector containing mGluR1a was cotransfected with the EGFP-N1 (Clontech) marker-containing vector at a ratio of 10:1 using SuperFect reagent (Qiagen). EGFP-expressing cells were identified with excitation at 480 nm and emission at 510 nm. Measurement of Fura-2 fluorescence was performed as described previously (41, 42). Intensities of fluorescent light emission at 510 nm, using excitation at 340 and 380 nm, were converted to Ca2+ concentrations as described previously (42). Whole-cell recording of Ba2+ currents was conducted as described previously (39). An external solution contained (mM) 3 BaCl2, 155 tetraethylammonium chloride, 10 HEPES, and 10 creatine, adjusted to pH 7.2. A pipette solution contained (mM) 85 cesium aspartate, 40 CsCl, 4 MgCl2, 5 EGTA, 2 ATP-Mg, 5 HEPES, and 10 creatine phosphate, adjusted to pH 7.2. In some experiments, cells were preincubated with staurosporin or U73122
[GenBank]
for
5 min by adding 500 nM staurosporin (Sigma) and 1 µM U73122
[GenBank]
(Calbiochem) in the pipette solution or pretreated with 300 ng/ml PTX (Seikagaku) for 24 h. The current-voltage relationship was obtained by holding the membrane potential at 80 mV and then delivering a depolarizing pulse for 30 ms in a stepwise manner with 10-mV increments from 50 to 60 mV.
Calcium Imaging of Cultured PNsPrimary culture and calcium imaging of PNs were performed as described previously (35). Briefly, PNs were loaded with Fura-2AM (10 µM, Molecular Probes) for 30 min at 37 °C. Recording was made with excitation at 340 and 380 nm for 250 ms and emission at 510 nm. Cells were first stimulated with 25 mM KCl in the culture medium containing 1 µM tetrodotoxin (Sigma), 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (Tocris), and 20 µM bicuculline (Sigma) (Medium A). The medium was replaced with the normal culture medium and incubated in the presence and absence of 50 µM 3,5-dihydroxyphenylglycine (DHPG) (Tocris), 50 µM DHPG plus 10 µM 7-hydroxyiminocyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (Tocris) and 100 nM
-agatoxin IVA (Peptide Institute). The incubation was carried out at 37 °C for 10 min in a CO2 incubator with 5% CO2/95% air. PNs were then stimulated with 25 mM KCl in Medium A with and without the above agonists or antagonists at 24 °C.
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RESULTS
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Colocalization of mGluR1a and Cav2.1 in Cerebellar PNsIt has been shown that mGluR1a is abundantly expressed in PNs (24, 43), whereas Cav2.1 is distributed at both granule cells and PNs (19, 21, 22). We examined codistribution of mGluR1a and Cav2.1 by immunostaining of mouse cerebellar sections with anti-mGluR1a and anti-Cav2.1 antibodies (Fig. 1, A and B). Strong immunoreactive signals of mGluR1a and Cav2.1 were observed throughout the molecular layer of the cerebellum (Fig. 1, A and B). Neither mGluR1a nor Cav2.1 immunoreactivity was seen in cerebellar sections of mGluR1 knockout mice (32, 36) and Cav2.1 knockout mice,2 respectively, with the antibodies we used. Double immunostaining of cerebellar sections was carried out with anti-mGluR1 and anti-Cav2.1 antibodies. This analysis showed that intense immunoreactive signals of the two proteins overlapped throughout the molecular layer but both signals were weak at the Purkinje cell layer (Fig. 1, CE). We further characterized mGluR1 and Cav2.1 immunoreactivities in cultured PNs, which extended long dendritic branches during continuing culture. Both immunoreactivities showed punctuate labeling patterns and were colocalized at the dendrites of cultured PNs (Fig. 1F). Immunoreactive signals of Cav2.1 were also seen on soma of cultured PNs, but mGluR1a was poorly immunostained on this region (Fig. 1G). Therefore, in both cerebellar sections and cultured PNs, Cav2.1 and mGluR1a immunoreactivities are abundantly localized at dendritic trees of PNs.

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FIG. 1. Colocalization of mGluR1 and Cav2.1 in the cerebellum. A and B, mouse cerebellar sections were immunostained with anti-mGluR1a and anti-Cav2.1 antibodies. Strong immunoreactive signals of both proteins were seen in the molecular layer of the cerebellum. CE, confocal images of double immunostaining with anti-mGluR1 and anti-Cav2.1 antibodies of mouse cerebellar cortical sections. Intense immunoreactive signals of both proteins were seen in the molecular layer (Mo), whereas these signals were weak in the Purkinje cell layer (Pu) and were lacking in somata of molecular layer interneurons. Gr, granular layer. F and G, merged images of mGluR1 (green) and Cav2.1 (red) immunoreactivities in cultured PNs, showing colocalization of these proteins at dendrites but not at soma of cultured PNs. Scale bars, 500 µm in A and B and 20 µm in CG.
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Interaction between mGluR1a and Cav2.1 in the Rat Brain and Transiently Transfected COS-7 CellsWe examined the possible protein assembly of mGluR1a and Cav2.1 in vivo by immunoprecipitation of solubilized rat brain membrane fractions. Solubilized membrane fractions were immunoprecipitated with anti-Cav2.1 and anti-mGluR1a antibodies, and the immunoprecipitates were immunoblotted with anti-mGluR1a and anti-Cav2.1 antibodies, respectively. The result showed that anti-Cav2.1 and anti-mGluR1a antibodies mutually coimmunoprecipitated mGluR1a and Cav2.1, respectively (Fig. 2, A and B). In control, neither of them was detected in immunoprecipitates with nonimmunized control IgG (Fig. 2, A and B).

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FIG. 2. Coimmunoprecipitation of mGluR1a and Cav2.1 from the rat brain and transiently expressing COS-7 cells. A, solubilized rat brain membrane fractions were immunoprecipitated with either anti-Cav2.1 antibody or control nonimmunized IgG, followed by immunoblotting with anti-mGluR1a antibody. mGluR1a was coimmunoprecipitated with anti-Cav2.1 antibody. B, solubilized rat brain membrane fractions were immunoprecipitated with either anti-mGluR1a antibody or control nonimmunized IgG, followed by immunoblotting with anti-Cav2.1 antibody. Cav2.1 was coimmunoprecipitated with anti-mGluR1a antibody. In A and B, inputs show 1 of 10 of extracts used for immunoprecipitation. C and D, COS-7 cells were transfected with mGluR1a, Cav2.1-Myc-His, or both. Cell lysates were immunoprecipitated with either anti-Myc antibody (C), anti-mGluR1a antibody (D), or control IgG, followed by immunoblotting with anti-mGluR1a (C) and anti-His antibodies (D). Inputs (lanes 14) show 1 of 20 of extracts used for immunoprecipitation.
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The complex formation of mGluR1a and Cav2.1 was further examined by cotransfection of mGluR1a and Myc/His-tagged Cav2.1 (Cav2.1-Myc-His) into COS-7 cells. COS-7 cell lysates were immunoprecipitated with anti-Myc and anti-mGluR1a antibodies, and the immunoprecipitates were immunoblotted with anti-mGluR1a and anti-His antibodies, respectively. Anti-Myc and anti-mGluR1a antibodies coimmunoprecipitated mGluR1a and Cav2.1-Myc-His in lysates of transiently cotransfected COS-7 cells, respectively (Fig. 2, C and D, lane 6). This coimmunoprecipitation was not seen in cells transfected with either mGluR1a or Cav2.1-Myc-His alone (Fig. 2, C and D, lanes 5 and 7) nor from cotransfected cell lysates precipitated with nonimmunized control IgG (Fig. 2, C and D, lane 8). These results indicate that mGluR1a and Cav2.1 form a protein complex in both rat brain and heterologously expressing cells.
Direct Interaction between mGluR1a and Cav2.1To examine the direct protein-protein interaction between mGluR1a and Cav2.1, we dissected the intracellular domains of Cav2.1 and then tested their ability to bind to the whole carboxyl-terminal domain of mGluR1a by in vitro pull-down assay. The amino-terminal domain, the intracellular loops, and the carboxyl-terminal domain of Cav2.1 were fused to GST (Fig. 3A), expressed in E. coli, and purified. The whole intracellular carboxyl-terminal domain of mGluR1a was fused to His/FLAG-tagged EGFP (HFG-1CT), expressed in insect cells, and purified (Fig. 3B). The various GST fusion proteins were immobilized on glutathione-Sepharose 4B beads and incubated with HFG-1CT. Bound proteins were eluted and immunoblotted with anti-His antibody (Fig. 3, A and C). This analysis showed that the carboxyl-terminal domain of mGluR1a not only bound strongly to the carboxyl-terminal portion of Cav2.1 (amino acid residues 18212424; GST-CavCT) but also weakly to the intracellular loop IIIII (residues 7151036; GST-CavIIIII-1) of Cav2.1 (Fig. 3C). We further defined the interaction site of the intracellular carboxyl-terminal region of Cav2.1 (Fig. 3, A and D). The GST fusion protein containing residues of 19752424 of Cav2.1 (GST-CTa) bound to HFG-1CT (Fig. 3D). Two amino-terminal portions covering residues 18212024 and 18212164 (GST-CTb and GST-CTc) failed to bind to HFG-1CT (Fig. 3D), suggesting that the remaining carboxyl-terminal domain of Cav2.1 is a likely candidate for interaction with mGluR1a. To confirm this observation, we attempted to express GSTs fused to the carboxyl-terminal portions of residues 20252424 or 21162424 of Cav2.1 in E. coli. However, these fusion proteins could not be detected in E. coli lysates, probably due to their rapid degradation. Although we have not proceeded further with this analysis, it is clear from the data so far attained that the intracellular carboxyl-terminal region of Cav2.1 directly interacts with the intracellular region of mGluR1a.

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FIG. 3. Direct interaction between mGluR1a and Cav2.1. The structural diagrams of the intracellular regions of rabbit Cav2.1 and rat mGluR1a tested for GST pull-down assays are shown in A and B, respectively. C, GST and GST fusion proteins containing the intracellular fragments of Cav2.1 were immobilized on glutathione-Sepharose 4B beads and incubated with the whole intracellular carboxyl-terminal domain of mGluR1a fused to His/FLAG-tagged EGFP (HFG-1CT); 1 µg each of purified GST, GST fusion proteins, and HFG-1CT was used for all experiments. Bound proteins were immunoblotted with anti-His antibody. D, purified GST and GST fusion proteins containing four different carboxyl-terminal regions of Cav2.1 (1 µg each) were analyzed as in C. E, purified GST and GST fusion proteins containing CavCT (1 µg each) were immobilized on glutathione-Sepharose 4B beads and incubated with His-tagged thioredoxin fused to four different carboxyl-terminal regions of mGluR1a (1 µg each). Bound proteins were immunoblotted with anti-His antibody. Inputs show equivalent amounts of the fusion proteins used for GST pull-down assays in CE.
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The binding site of mGluR1a was also examined by dividing its carboxyl-terminal portion into four regions, each fused to His-tagged thioredoxin (HT-1CT-1 to HT-1CT-4) (Fig. 3B). The four HT-1CT proteins were expressed in E. coli, purified, and incubated with glutathione-Sepharose 4B beads attached with GST-CavCT. Bound proteins were immunoblotted with anti-His antibody (Fig. 3E). This analysis showed that the carboxyl-terminal region close to the seventh transmembrane domain of mGluR1a (residues 841932) is responsible for a strong interaction with the carboxyl-terminal domain of Cav2.1 (Fig. 3E). The interaction between loop IIIII of Cav2.1 and the carboxyl-terminal domain of mGluR1a was not of sufficient strength (Fig. 3C) to allow further characterization of the interacting domain of mGluR1a. Collectively, the results indicate that mGluR1a and Cav2.1 form a complex through direct protein-protein interactions between their intracellular domains.
Ligand-independent Inhibition of Cav2.1 by Coexpression of mGluR1aWe examined the functional coupling of mGluR1a and Cav2.1 in heterologously transfected HEK 293 cells. In HEK 293 cells, neither P/Q-type channel nor mGluR1 responses were detected (data not shown). A cell line of HEK 293 cells permanently expressing Cav2.1 together with auxiliary
2/
and
1 subunits (HEK-Cav) was established (39). The HEK-Cav cells were then transiently cotransfected with the mGluR1a or the control vector. Cav2.1-mediated Ca2+ influx was evoked by depolarization with high KCl and measured with calcium imaging. Even when the mGluR1a agonist was not added, cotransfection of mGluR1a significantly inhibited a depolarization-induced [Ca2+]i increase as compared with that of control cells (Fig. 4A). This inhibition was indeed ligand-independent, because an mGluR1 antagonist, CPCCOEt (10 µM), had no effect on the mGluR1a-mediated inhibition of [Ca2+]i increase (Fig. 4B). The inhibitory effect of mGluR1a was further confirmed by whole-cell recording of Cav2.1-mediated Ba2+ currents in HEK-Cav cells. When membrane potentials were depolarized from a holding potential of 80 mV to positive potentials, HEK-Cav cells gave rise to inward Ba2+ currents in the Ba2+-containing external solution (Fig. 4C, left panel). The current-voltage relationship of Ba2+ currents showed a bell-shape characteristic of P/Q-type channels (39) (Fig. 4C, right panel). Comparison of mGluR1a-expressing and control HEK-Cav cells showed that Ba2+ currents were significantly inhibited by coexpression of mGluR1a at all ranges of depolarization potentials (n = 19 for mGluR1a and n = 18 for control; p < 0.001, repeated measures analysis of variance) (Fig. 4C). When mGluR1a-expressing HEK-Cav cells were pretreated with the Gi/Go inhibitor PTX (300 ng/ml), the protein kinase C inhibitor staurosporin (500 nM), or the phospholipase C inhibitor U73122
[GenBank]
(1 µM), they failed to relieve the mGluR1a-mediated inhibition of Ba2+ currents (Fig. 4D and data not shown). This finding indicates that the ligand-independent inhibition of Cav2.1 channels is derived from expression of mGluR1a per se rather than involvement of G proteins or their downstream effectors.

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FIG. 4. Ligand-independent inhibition of the Cav2.1 activity by mGluR1a coexpression. A, time courses (left panel) and peak amplitudes (right panel) of [Ca2+]i increases evoked by 50 mM KCl in HEK-Cav cells transfected with the mGluR1a expression vector and the control vector. Data were averaged (n = 34 for mGluR1a and n = 27 for control). Columns and bars in the right panel represent mean ± S.E.; **, p < 0.01 (Student's t test). B, KCl-induced [Ca2+]i increases of mGluR1a-expressing HEK-Cav cells were measured in the presence and absence of CPCCOEt (10 µM). Columns and bars indicate mean ± S.E.; n = 15 for control and n = 19 for CPCCOEt; n.s., not significant. C, whole-cell recordings of Ba2+ currents of mGluR1a-expressing and control HEK-Cav cells were conducted by holding the membrane potential at 80 mV and then delivering a depolarizing pulse (as shown by an arrow) for 30 ms in a stepwise manner with 10-mV increments from 50 to 60 mV. Representative current traces are indicated in the left panel. Current-voltage relationships of Ba2+ currents of mGluR1a-expressing and control HEK-Cav cells are shown in the right panel. Ba2+ currents at each depolarization potential were averaged; n = 19 for mGluR1a and n = 18 for control. Circles and bars indicate mean ± S.E. D, Ba2+ currents elicited by a depolarizing pulse of 10 mV from the holding potential at 80 mV were measured by whole-cell recordings of control HEK-Cav cells (n = 5) and mGluR1a-expressing HEK-Cav cells with (n = 5) and without (n = 17) pretreatment of PTX (300 ng/ml) for 24 h. Columns and bars indicate mean ± S.E.; *, p < 0.05; n.s., not significant.
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Temporally Regulated Modulation of Cav2.1 Channels by mGluR1a ActivationWe next addressed whether ligand activation of mGluR1a affects depolarization-induced [Ca2+]i increase by calcium imaging. When mGluR1a was activated by application of group 1 mGluR-selective agonist, DHPG, 5 min prior to depolarization, the activation of mGluR1a caused strong inhibition of KCl-induced [Ca2+]i increase (Fig. 5A). mGluR1a thus inhibited the Cav2.1-mediated [Ca2+]i increase in both a ligand-independent and a ligand-dependent manner. The ligand-dependent inhibition was further confirmed by whole-cell recording of Ba2+ currents. Preincubation of these cells with glutamate (100 µM) significantly inhibited depolarization-inducedBa2+currents(Fig.5B).Furthermore,this ligand-dependent inhibition was at least partially relieved by pretreatment with PTX (300 ng/ml) (Fig. 5B). However, neither staurosporin (500 nM) nor U73122
[GenBank]
(1 µM) showed any suppressive effect on ligand-dependent inhibition of Ba2+ currents (Fig. 5B): percentages of inhibition of Ba2+ currents, relative to Ba2+ currents measured in the absence of glutamate, were 26.9 ± 2.8% for glutamate alone (n = 18), 12.1 ± 1.5% for glutamate plus PTX (n = 5; p < 0.05; Student's t test), 32.1 ± 10.0% for glutamate plus staurosporin (n = 5), and 36.0 ± 6.9% for glutamate plus U73122
[GenBank]
(n = 2). The membrane-delimited mGluR1a-Gi/Go signaling is thus most likely implicated in the ligand-dependent, mGluR1a-mediated inhibition of Cav2.1 channels.

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FIG. 5. [Ca2+]i responses evoked by depolarization and mGluR1 agonist in mGluR1a-expressing HEK-Cav cells. A, [Ca2+]i responses of mGluR1a-expressing HEK-Cav cells were measured by addition of 50 mM KCl alone (KCl) (n = 34), 50 mM KCl after pretreatment with 50 µM DHPG for 5 min (preDHPG + KCl) (n = 34), 50 µM DHPG alone (DHPG) (n = 34), and simultaneous addition of 50 µM DHPG and 50 mM KCl (DHPG + KCl) (n = 31). [Ca2+]i increases in each treatment were averaged. Time courses and peak amplitudes of [Ca2+]i increases are indicated in the left and right panels, respectively; a line below [Ca2+]i responses shows KCl and/or DHPG treatments. In the right panel, columns and bars indicate mean ± S.E.; **, p < 0.01; *, p < 0.05 (Student's t test). B, mGluR1a-expressing HEK-Cav cells were preincubated with or without 100 µM glutamate for 30 s in the presence and absence of PTX (300 ng/ml), staurosporin (500 nM), and U73122
[GenBank]
(1 µM). Ba2+ currents were evoked by a depolarizing pulse of 10 mV (as shown by an arrow) for 50 ms from the holding potential at 80 mV and measured by whole-cell recording.
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Application of DHPG alone increased [Ca2+]i by mobilizing Ca2+ from intracellular Ca2+ stores (Fig. 5A) (7). When mGluR1a and Cav2.1 channels were simultaneously activated by DHPG and high KCl, [Ca2+]i was markedly increased as compared with a [Ca2+]i increase elicited by either high KCl or DHPG alone (Fig. 5A). This finding indicates that mGluR1a in association with Cav2.1 channels plays an important role in a temporally regulated [Ca2+]i increase.
Modulation of P-type Channel Activity by mGluR1 Agonists in Cultured PNsWe addressed whether the coupling of mGluR1a and Cav2.1 modulates [Ca2+]i responses in neurons by calcium imaging of cultured PNs. Depolarization of cultured PNs with high KCl increased [Ca2+]i at dendrites and soma of PNs (Fig. 6, A and B). The [Ca2+]i increases were blocked at both dendrites and soma by the P-type channel blocker
-agatoxin IVA (n = 3) (Fig. 6C). This finding was consistent with previous pharmacological and electrophysiological studies indicating the predominant role of P-type Ca2+ channels in Ca2+ permeation of PNs (5, 44). The mGluR1-mediated modulation of P-type channels was then examined by addition of DHPG (50 µM) before and at the time when P-type channels were activated by high KCl. Extents of P-type channel activation were unchanged when PNs were depolarized twice with a 10-min interval by high KCl (n = 5) (Fig. 6C). When DHPG was added during a 10-min interval, it significantly inhibited the P-type channel-mediated [Ca2+]i increase at dendrites of PNs (Fig. 6). Because mGluR5 was undetectable in PNs with both in situ hybridization and immunohistochemistry (8, 45), the effect of DHPG was attributable to the inhibitory function of mGluR1 on P-type Ca2+ channels. Furthermore, the inhibition by DHPG was blocked by concomitant incubation of an mGluR1 antagonist CPCCOEt (10 µM) (data not shown). mGluR1 is thus responsible for inhibition of the P-type channel activity.
The simultaneous stimulation of PNs with DHPG and high KCl slightly but significantly increased KCl-induced [Ca2+]i at dendrites of PNs (Fig. 6C). Furthermore, reflecting the predominant localization of mGluR1 at dendrites over soma of PNs (Fig. 1, F and G), both stimulatory and inhibitory effects of mGluR1 on [Ca2+]i increase were confined to dendrites of PNs (Fig. 6). This finding indicates that the functional coupling of mGluR1 and Cav2.1 plays an important role in spatiotemporal modulation of P-type channels at PNs.
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DISCUSSION
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This investigation has demonstrated that mGluR1a and Cav2.1 are colocalized in dendrites of PNs and form the heteromeric assembly in both the rat brain and heterologously expressing COS-7 cells. This assembly occurs through the direct interaction between the carboxyl-terminal region of Cav2.1 and the cytoplasmic region close to the seventh transmembrane region of mGluR1a. The binding region of mGluR1a is relatively hydrophilic (23) and possesses Ca2+/calmodulin binding motifs (46). This region has been shown not only to interact with Ca2+/calmodulin and E3 ubiquitin ligase Siah (46) but also to be essential for coupling to the G protein G
q (47). The discrete binding region of Cav2.1 remains to be determined, but truncation analysis suggests that the distal portion of the intracellular carboxyl-terminal region of Cav2.1 is a likely candidate for interaction with mGluR1a. Sequence data analysis of this region has shown the presence of several sequence motifs, including proline-rich (residues 23082394), arginine-rich (residues 22022404), and histidine-rich sequences (residues 22162227), which have been implicated in protein-protein interaction. Recently, evidence is accumulated indicating that several transmembrane receptors and ion channels form heteromers through direct protein-protein interaction. The
-aminobutyric acid, type A receptor channels selectively complex with dopamine D5 receptor through the direct interaction between the D5 receptor carboxyl-terminal domain and the intracellular loop of the
-aminobutyric acid, type A,
2 receptor subunit (48). This interaction confers mutual inhibition in a ligand-dependent manner (48). The sigma receptor also binds to the Kv1.4 and Kv1.5 voltage-gated K+ channels and inhibits the K+ channel activity in different ways in the presence and absence of sigma receptor ligands (49). The
2 adrenergic receptor not only associates with L-type Ca2+ channels but also forms a multimolecular signaling complex, thus affording highly localized signal transduction from the receptor to the Ca2+ channels (50). mGluR1 binds to key scaffold proteins, homers and tamalin, which form protein complexes with multiple synaptic scaffold proteins and intracellular signaling molecules (38, 5154). The assembly of mGluR1, Cav2.1, and signaling molecules may thus provide mechanisms that ensure specific and efficient signaling in mGluR1-mediated glutamatergic transmission.
The inhibition of P/Q-type Ca2+ channels by group 1 mGluRs has been reported in several neuronal cells as well as heterologously expressing cells (30, 31, 55). This investigation has indicated that mGluR1a inhibits Cav2.1 Ca2+ channels in both ligand-dependent and ligand-independent manners, which are mediated by the mechanisms of the membrane-delimited Gi/Go coupling and the physical coupling between the two proteins, respectively. In cultured PNs, Cav2.1 channels are distributed at both soma and dendrites, but mGluR1 is mainly localized at dendrites. Reflecting the specialized localization of mGluR1, the pre-activation of mGluR1 preferentially inhibits P-type channels at the dendrites of cultured PNs. This dendritic inhibition of P/Q-type Ca2+ channels may commonly occur in other neurons, because the similar inhibition of P/Q-type Ca2+ channels by mGluRs has been reported in isolated hippocampal dendritic segments (56). The physical interaction between mGluR1a and Cav2.1 Ca2+ channels would thus contribute to spatially restricted modulation of Ca2+ influx by activation of mGluR1a.
This investigation has demonstrated that the conjunctive activation of mGluR1a and Cav2.1 augments [Ca2+]i increase in both heterologously expressing cells and cultured PNs. In the cerebellar slices, coincident stimulation of parallel fibers (PFs) and a climbing fiber (CF) evokes [Ca2+]i increases at dendritic spines of PNs, which are much larger than the sum of [Ca2+]i increases induced by separate stimulation of PFs and CF (27). Because IP3 receptors are synergistically coactivated by IP3 and Ca2+ (57), IP3 receptors have been proposed to be a candidate coincident detector to induce a supralinear [Ca2+]i increase in PNs (2, 2729, 58, 59). Our data showed no such supralinear [Ca2+]i increase resulting from simultaneous activation of mGluR1a and Cav2.1. This absence of supralinearity may be due to the use of the high affinity Ca2+ indicator Fura-2, which chelates the cytosolic Ca2+ and reduces Ca2+ signaling (59). Importantly, our data have indicated that the simultaneous activation of mGluR1a and Cav2.1 channels, in contrast to mGluR1a pre-activation, enhances [Ca2+]i increase in both cultured PNs and heterologously expressing cells. This different modulation of the Cav2.1 channels could provide a temporal window in [Ca2+]i responses of PNs. Furthermore, it has been shown that the scaffold protein homer links mGluR1a to IP3 receptors and ensures efficient Ca2+ signal transmission from mGluR1a to IP3 receptors in PNs (53). It is thus possible that the close proximity of mGluR1a, Cav2.1, and IP3 receptors may help IP3 receptors sensing changes of Ca2+ influx derived from Cav2.1 Ca2+ channels. Numerous lines of evidence have indicated that an enhanced [Ca2+]i increase by the coincident activation of PFs and CF is essential for long lasting depression in PF synaptic transmission onto PNs (2729). It is therefore tempting to speculate that the temporally regulated modulation of Cav2.1 Ca2+ channels by mGluR1a contributes to integration of synaptic inputs onto PNs.
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FOOTNOTES
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* This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. 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. 
** To whom correspondence should be addressed. Tel.: 81-75-753-4437; Fax: 81-75-753-4404; E-mail: snakanis{at}phy.med.kyoto-u.ac.jp.
1 The abbreviations used are: VSCC, voltage-sensitive Ca2+ channel; mGluR, metabotropic glutamate receptor; IP3, inositol 1,4,5-trisphosphate; PN, Purkinje neuron; GST, glutathione S-transferase; EGFP, enhanced green fluorescent protein; DHPG, 3,5-dihydroxyphenylglycine; CPCCOEt, 7-hydroxyiminocyclopropa[b]chromen-1a-carboxylate ethyl ester; PTX, pertussis toxin; E3, ubiquitin-protein isopeptide ligase; PFs, parallel fibers; CFs, climbing fibers. 
2 A. Kulik and R. Shigemoto, personal communication. 
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ACKNOWLEDGMENTS
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We thank Nagahiro Minato, Yoshimasa Tanaka, Hisato Jingami, and Yoshimi Shimada for technical assistance; Ryuichi Shigemoto for providing mGluR antibodies and helpful discussion; and Peter Sillevis Smitt for providing anti-mGluR antibodies.
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