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The Journal of Neuroscience, November 1, 2000, 20(21):7896-7904

Selective Blockade of P/Q-Type Calcium Channels by the Metabotropic Glutamate Receptor Type 7 Involves a Phospholipase C Pathway in Neurons

Julie Perroy¹, Laurent Prezeau¹, Michel De Waard², Ryuichi Shigemoto³, Joel Bockaert¹, and Laurent Fagni¹

¹ Centre National de la Recherche Scientifique, Unité Propre de Recherche 9023, Centre CNRS-INSERM de Pharmacologie et d'Endocrinologie, 34000 Montpellier, France, ² Institut National de la Santé et de la Recherche Médicale, U-464, Faculté de Médecine Nord, 13916 Marseilles, France, and ³ Laboratory of Cerebral Structure, National Institute for Physiological Sciences, CREST Japan Science and Technology Corporation, Myodaiji, Okazaki 444-8585, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca²⁺ channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca²⁺ channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca²⁺ channels. The effect was mimicked by GTPS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein o subunit, indicating the involvement of a G_o-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein subunits and the 1A Ca²⁺ channel subunit, but was abolished by quenching subunits with specific intracellular peptides. Intracellular dialysis of G-protein subunits did not mimic the action of mGluR7, suggesting that both G-protein and o subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein with o subunits was required. The Ca²⁺ chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP₃) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP₃ formation. These results indicated that IP₃-mediated intracellular Ca²⁺ release was required for PKC-dependent inhibition of the Ca²⁺ channels. Possible control of synaptic transmission by the present mechanisms is discussed.

Key words: mGluR7; Ca²⁺ channels; G-protein; PLC; cerebellar granule cells; transfection

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The physiological actions of the neurotransmitter glutamate are mediated by ionotropic and metabotropic receptors (Nakanishi, 1992). Eight genes encoding mGluRs have been identified and classified into three groups. mGluR1 and mGluR5 belong to group I and activate phospholipase C (PLC) through stimulation of a G_q protein, in heterologous and homologous systems (Conn and Pin, 1997). The group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) mGluRs are coupled to G_i/o protein in neuron (Prezeau et al., 1994) and heterologous expressing cells (Conn and Pin, 1997). These receptors are widely distributed throughout the mammalian brain (Kinzie et al., 1995; Ohishi et al., 1995; Bradley et al., 1996; Kinoshita et al., 1998), but the mGluR7 subtype displays peculiar properties in that it is almost exclusively localized at presynaptic sites (Shigemoto et al., 1996, 1997; Kinzie et al., 1997). Because of a lack of specific pharmacology, functional discrimination between mGluR7 and the other group III mGluR subtypes can only be achieved according to their different affinity for L-2-amino-4-phosphonobutyrate (L-AP-4), a selective group III mGluR agonist. Indeed the affinity of mGluR7 for L-AP-4 is clearly lower (EC₅₀ = 160-500 µM; Okamoto et al., 1994; Saugstad et al., 1994) than that of mGluR4, 6, and 8 (EC₅₀ = 0.2-1.2, 0.9, and 0.06-0.60 µM, respectively; Pin et al., 1999).

In behavioral studies, young mGluR7 knock-out mice display deficits in the fear response and conditioned taste aversion, whereas the adult mutants develop lethal spontaneous epileptic seizures (Masugi et al., 1999). In vitro studies showed that mGluR7 stimulation mediates neuroprotective effects in cultured cerebellar granule cells by decreasing glutamate release (Lafon-Cazal et al., 1999a) and promotes excitotoxicity in cultured striatal neurons by inhibiting GABA release (Lafon-Cazal et al., 1999b). Group III mGluRs, presumably mGluR7, have been shown to inhibit glutamate autaptic currents in hippocampal neurons (O'Connor et al., 1999). These studies, together with those showing the presynaptic localization of the receptor in the murine adult brain, suggest that mGluR7 plays an important role in modulation and plasticity of synaptic transmission.

The mechanism by which mGluR7 may control neurotransmitter release is still unknown. Indeed, previous studies have shown that L-AP-4 inhibits high-threshold voltage-gated Ca²⁺ channels in various neuronal preparations (Trombley and Westbrook, 1992; Rothe et al., 1994; Choi and Lovinger, 1996; Takahashi et al., 1996; Shen and Slaughter, 1998). Nevertheless, in these studies, the maximal inhibitions were obtained for relatively low concentrations of L-AP-4 (<100 µM) that should have selectively activated group III mGluRs, but with the exception of mGluR7. Moreover, inhibition of adenylyl cyclase by mGluR7 has only been shown in heterologous expression systems (Okamoto et al., 1994; Saugstad et al., 1994), and to our knowledge there is no clear study precluding that a different mechanism may function in neurons. Therefore, in the present study we investigated whether mGluR7 could modulate specific Ca²⁺ channel subtypes in cultured cerebellar granule cells and which coupling mechanism could be involved in this effect. We found that the receptor selectively inhibited P/Q-type Ca²⁺ channels by activating a G_o-like protein and, unexpectedly, through a PLC-dependent pathway.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Primary cultures of cerebellar cells were prepared as previously described (Van Vliet et al., 1989). Briefly, 1-week-old newborn mice were decapitated and cerebellum-dissected. The tissue was then gently triturated using fire-polished Pasteur pipettes, and the homogenate was centrifuged at 500 rpm. The pellet was resuspended and plated in tissue culture dishes previously coated with poly-L-ornithine. Cells were maintained in a 1:1 mixture of DMEM and F-12 nutrient (Life Technologies, Gaithersburg, MD), supplemented with glucose (30 mM), glutamine (2 mM), sodium bicarbonate (3 mM) and HEPES buffer (5 mM), decomplemented fetal calf serum (10%), and 25 mM KCl to improve neuronal survival. One-week-old cultures contained 125 × 10³ cells/cm².

Plasmids and transfection. The N-terminal epitope-tagged mGluR7a receptor was constructed as follows. The Myc epitope was inserted in the extracellular domain, immediately downstream from the signal peptide. We used a mGluR5a-containing plasmid (pRKG5a-N-Myc) as the starting vector, in which the signal peptide was followed by the Myc-coding sequence at the N terminus of the protein, and then by a MluI site (Ango et al., 1999). The mGluR7a-coding sequence (except the signal peptide) was introduced into this vector using the MluI site and XbaI (3' of the coding sequence), by following two steps: first we used a PCR and the oligonucleotide S, creating an Mlu I site in frame with the coding sequence of the vector (5'-gccAcgcgtatgtacgccccgcac-3'), and the oligonucleotide AS containing the XhoI site present in mGluR7 (5'-ttttctagaggaaggaatcaggcgggacca-3'); second the fragment XhoI-XbaI was inserted by classical subcloning. The sequence was verified by sequencing. The resulting plasmid (pRKG7a-N-Myc) was referred as Myc-mGluR7. Functional coupling of Myc-mGluR7 was verified in human embryonic kidney 293 cells according to the protocol described elsewhere (Parmentier et al., 1998).

Immediately before plating, or 24 hr after plating, cerebellar cultures were transfected with the Myc-mGluR7 expression plasmid for immunocytochemical experiments or cotransfected with the transfection marker, green fluorescent protein (GFP)-containing plasmid, pEGFP-N1 (Clontech, Palo, Alto, CA), and non epitope-tagged mGluR7, for electrophysiological recordings, by using Transfast (Promega, Madison, WI), as described elsewhere (Ango et al., 1999).

Immunocytochemistry. Cultured cerebellar granule cells were fixed in a 4% paraformaldehyde and 0.1 M glucose-containing PBS solution. The culture was permeabilized with 0.05% Triton X-100, and fluorescent immunolabeling of native mGluR7 was performed by using a previously characterized anti-mGluR7a/b primary antibody (Shigemoto et al., 1996, 1997). The presence of Myc-mGluR7 protein at the cell surface of cultured neurons was examined in nonpermeabilized cerebellar cultures exposed to a monoclonal mouse anti-Myc primary antibody (a gift from B. Mouillac) diluted at 1:300 in a PBS-gelatin (0.2%) solution. After overnight incubation in the presence of either one of these primary antibodies at room temperature, cells were then rinsed and exposed to a goat Texas Red-conjugated anti-rabbit IgG secondary antibody or to a goat Texas Red-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch, West Grove, PA; 1:1000 dilution), for 2 hr at room temperature. Then cells were rinsed again with PBS and mounted on glass coverslips for observation on an Axiophot 2 Zeiss microscope.

Electrophysiology. Whole-cell patch-clamp Ba²⁺ currents were recorded at room temperature from GFP and mGluR7 cotransfected cerebellar granule cells, after 9 ± 1 days in vitro as previously described (Ango et al., 1999). The bathing medium contained (in mM): BaCl₂ 20, HEPES 10, tetraethylammonium acetate 10, TTX 3 × 10⁴, glucose 10, sodium acetate 120, and MK801 1 × 10³, adjusted to pH 7.4 with NaOH and 330 mOsm with sodium acetate. Drug solutions were prepared in this bathing medium, and the pH was adjusted to 7.4. The NMDA receptor-channel blocker MK-801 (1 µM) was added to all the solutions to avoid activation of this receptor by the D isoform of D,L-AP-4 (our unpublished observation). Patch pipettes were made from borosilicate glass, coated with Sylgard, and the tip was fire-polished. Pipettes had resistances of 3-5 M when filled with the following internal solution (in mM): Cs-acetate 100, MgCl₂ 2, HEPES 10, glucose 15, CsCl 20, EGTA 20, Na₂ATP 2, and cAMP 1, adjusted to pH 7.2 with CsOH and 300 mOsm with CsOH. In some experiments, intracellular EGTA was replaced by BAPTA.

Ba²⁺ currents were evoked by voltage-clamp pulses of 500 msec duration, from a holding potential of 80 mV to a test potential of 0 mV. Voltage pulses were applied at a rate of 0.1 Hz. Current signals were recorded with an Axopatch 200 amplifier, filtered at 1 kHz with an 8-pole Bessel filter, and sampled at 3 kHz on a Pentium II personal computer. Linear leak and capacitive currents were digitally subtracted from records before analysis by using the P/N procedure of the pClamp6 software of Axon Instruments (Foster City, CA). Analyses were performed by using the Clampfit subprogram of pClamp6. Ba²⁺ currents were measured at their peak amplitude and expressed as mean ± SEM of the indicated number (n) of experiments. In experiments in which neurons were dialyzed with compounds, current measurements were started at least 5 min after breaking the patch.

The intracellular I-II loop of the 1A Ca²⁺ channel subunit, which contained the binding site of G-protein subunits and P/Q-type Ca²⁺ channel  subunit, was generated in the laboratory, according to the following procedure. The 68-mer peptide, corresponding to the sequence from 360 to 427 of the 1A (BI-2) subunit, was synthesized by the solid-phase method (Merrifield, 1986) by means of an automated peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA). The peptide chain was assembled by a double-coupling strategy using Fmoc amino acid hydroxybenzotrioazol active esters. The crude peptide was purified to homogeneity by C18 reversed-phase HPLC and characterized by amino acid analysis after acidolysis, Edman sequencing, and mass spectrometry. The experimental values obtained were all in agreement with the theoretically deduced values.

Measurement of inositol phosphate accumulation. The procedure we used to measure inositol triphosphate (IP₃) accumulation in neurons was adapted from one previously described (Blahos et al., 1998). One-week-old cerebellar granule cell cultures were incubated for 14 hr in culture medium containing 2 µCi/ml myo-(³H)inositol (23.4 Ci/mol) (NEN, Paris, France). Cells were then washed three times and incubated for 1 hr at 37°C, in 1 ml of HEPES saline buffer (in mM: NaCl 146, KCl 4.2, MgCl₂ 0.5, and HEPES 20, glucose 0.1%, pH 7.4) supplemented with 1 U/ml glutamate pyruvate transaminase (Boehringer Mannheim, Meylan, France) and 2 mM pyruvate (Sigma, Lisle d'Abeau, France). Cells were then washed again with the same buffer, and LiCl was added to a final concentration of 10 mM. The agonist was applied 15 min later and left for 5 min. The reaction was stopped by replacing the incubation medium with 0.5 ml of perchloric acid (5%) on ice. Supernatants were recovered, and IPs were purified on Dowex columns (Berridge et al., 1983). Total radioactivity remaining in the membrane fraction was counted after treatment with 10% Triton X-100 and 0.1 N NaOH for 30 min and used as a standard. Results were expressed as the ratio of [³H]IP production over radioactivity present in the membranes. Experiments were performed in triplicates for statistical analyses.

Materials. L-AP-4, Dihydroxy-phenyl-glycine (DHPG), and MK-801 were purchased from Tocris Cockson. PTX, Nimodipine, GF109203X, U73122, and U73343 were purchased from Research Biochemicals (Natick, MA). -Agatoxin-IVA and -Conotoxin-GVIA were from Alomone Labs (Jerusalem, Israel). The G-protein subunits purified from bovine brain were from Calbiochem. The inhibitor peptide of the catalytic subunit and the competitive inhibitor of the regulatory subunit of protein kinase A, protein kinase A inhibitor peptide (PKI), and Rp-cAMPS respectively, were also from Calbiochem. GTPS was from Sigma, and PDBu and PMA were from Fluka. An antibody raised against the G_o-protein was a generous gift from V. Homburger. This antibody has been previously shown to specifically recognize the G-protein o but not i subunit (Lledo et al., 1992). The pcDNA3-CD8-ARK plasmid, which was composed of the CD8 antigen membrane receptor and a domain containing the G-protein subunit-binding site of ARK, was a generous gift from Dr. J. Lang. The pEGFP-N1 expression plasmid was purchased from Clontech.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Absence of native mGluR7 expression in the soma of cultured cerebellar granule cells

Immunolabeling of native mGluR7 in permeabilized cerebellar granule cells revealed a somatic exclusion and a neuritic punctate pattern of distribution of the receptor (Fig. 1A). D,L-AP-4, up to 1 mM concentration, did not significantly alter the whole-cell Ba²⁺ current of neurons transfected (Fig. 2A) or not (data not shown) with GFP alone (control). Together these results indicated that the native mGluR7 was absent at the surface of the soma of cultured cerebellar granule cells. Therefore these neurons were potentially a good model to study the effect of transfected mGluR7 on somatic Ca²⁺ channels in these neurons, providing that the transfected receptor would be expressed at the cell body membrane.

View larger version (91K): [in this window] [in a new window]   Figure 1.   Localization of native and transfected mGluR7 in cultured cerebellar granule cells. A, Native mGluR7 immunolabeling in permeabilized cultured cerebellar granule cells. B, Nonpermeabilized cultured cerebellar granule cell transfected with the Myc-mGluR7 expression plasmid and labeled with an anti-Myc antibody. Note the presence of neuritic clusters in A and B and presence of somatic immunolabeling only in B.

View larger version (38K): [in this window] [in a new window]   Figure 2.   Inhibitory effect of D,L-AP-4 on Ba2+ currents in mGluR7-transfected cerebellar granule cells. A, Each bar of the histogram represents the mean (± SEM; n = 10 to 18) of fractional reduction of whole-cell Ba2+ current induced by D,L-AP-4 (500 µM) applied alone, in cultured cerebellar granule cells transfected with GFP alone, or cotransfected with GFP + mGluR7. Note that D,L-AP-4 alone inhibited Ba2+ currents only in cotransfected cells. B, Ba2+ currents recorded in a mGluR7-transfected cell, in the absence and presence of 10 µM, 100 µM, 500 µM, or 1 mM D,L-AP-4. Please note the absence of change in activation kinetics in the presence of the agonist. C, D, Activation (C) and inactivation (D) curves of whole-cell Ba2+ currents obtained from two different granule cells, in the absence (control) and presence of D,L-AP-4 (500 µM). Similar results were obtained from five other cells. E, Time course and concentration-dependent effect of D,L-AP-4 on Ba2+ currents in a mGluR7-transfected cerebellar granule cell. F, Inhibitory effects of -Agatoxin-IVA (250 nM), -Conotoxin-GVIA (1 µM), and nimodipine (1 µM) on Ba2+ currents obtained in nontransfected cultured cerebellar granule cells or transfected with GFP alone or cotransfected with GFP + mGluR7. Each bar of the histogram represents the mean (± SEM) of at least seven experiments. G, Inhibitory effects of -Agatoxin-IVA (250 nM), -Conotoxin-GVIA (1 µM), and nimodipine (1 µM) on Ba2+ currents obtained in the presence of D,L-AP-4 (500 µM), in cultured cerebellar granule cells transfected with GFP alone or cotransfected with GFP + mGluR7. Each bar of histogram represents the mean (± SEM) of at least 10 experiments. Note that the percentage of Ba2+ current inhibited by each toxin was similar in control and cotransfected cells, except for -Agatoxin-IVA, which was ineffective only in cotransfected cells.

Selective inhibition of P/Q-type Ba²⁺ current by transfected mGluR7 at the soma of cultured cerebellar granule cells

Eight to ten days after transfection of the Myc-mGluR7 expression plasmid in cultured cerebellar granule cells, Myc immunostaining revealed the presence of both somatic and neuritic cell surface clusters of the recombinant receptors (Fig. 1B). In these transfected neurons, D,L-AP-4 (500 µM) decreased the amplitude of the total Ba²⁺ current, without significantly affecting its activation and inactivation kinetics (Fig. 2B). The effect started after a delay of ~20 sec, developed slowly, and reached a plateau over a 1 min 30 sec application of the agonist. This inhibition was accompanied by a slight but not significant modification of voltage-dependent activation (Fig. 2C) and no alteration of steady-state inactivation (Fig. 2D) properties of the current. The D,L-AP-4 effect was dose-dependent, the threshold effect being obtained for 100 µM, and the maximal effect (38% inhibition) for 500 µM concentrations (Fig. 2A,B). The current inhibition lasted for at least 10 min after washout of the agonist (Fig. 2E). This long-lasting D,L-AP-4-mediated inhibition of Ba²⁺ currents did not result from an agonist-independent run-down of the current, because the amount of inhibition was stable for several minutes during wash-out of the agonist (Fig. 2E). Moreover, no significant decrease of the current was observed over a period of 45 min, in the absence of D,L-AP-4 (data not shown).

In cultured cerebellar granule cells transfected with GFP alone (control), P/Q-type (-Agatoxin-IVA; 250 nM), N-type (-Conotoxin-GVIA; 1 µM), and L-type (nimodipine; 1 µM) Ca²⁺ channel blockers inhibited the whole-cell Ba²⁺ current by 41, 10, and 22%, respectively. The remaining 27% of total Ba²⁺ current were of the R-type. Similar results were obtained in cultured cerebellar granule cells cotransfected with GFP and mGluR7 or nontransfected cultured cerebellar granule cells (Fig. 2F). Therefore, our transfection procedure did not alter functional expression of native Ca²⁺ channels in the studied cells.

To determine which types of Ca²⁺ channels were inhibited by transfected mGluR7, D,L-AP-4 (500 µM) was applied first, followed by perfusion of different selective Ca²⁺ channel blockers, on neurons cotransfected with mGluR7 and GFP. After application of D,L-AP-4, the remaining Ba²⁺ current was not significantly affected by application of -Agatoxin-IVA (250 nM), but was further depressed by -Conotoxin-GVIA (1 µM) or nimodipine (1 µM), and in similar proportions as those obtained in control cells (transfected with GFP alone; Fig. 2G). It is worth noting that the fractional inhibition induced by D,L-AP-4 in cotransfected neurons (38%; Fig. 2A) was not significantly different from the fraction of -Agatoxin-IVA-sensitive current obtained in control neurons (41%; Fig. 2G).

In a second series of experiments, an initial application of a given channel blocker was immediately followed by application of D,L-AP-4 (500 µM). When -Agatoxin-IVA was applied first, the -Agatoxin-IVA-resistant Ba²⁺ current was not affected by subsequent perfusion of D,L-AP-4 (500 µM; 3 ± 1% inhibition, n = 7, Fig. 3A). On the other hand, when -Conotoxin-GVIA or nimodipine were applied first, the drug/toxin-insensitive Ba²⁺ current was further depressed by subsequent application of D,L-AP-4 (37 ± 2% inhibition, n = 5, after -Conotoxin-GVIA, Fig. 3B; 36 ± 4% inhibition, n = 5, after nimodipine, Fig. 3C; 36 ± 2% inhibition, n = 5, after -Conotoxin-GVIA and nimodipine, Fig. 3D). Finally, after coapplication of all three Ca²⁺ channel blockers, D,L-AP-4 did not further inhibit the remaining Ba²⁺ current (3 ± 2% inhibition, n = 4). After application of D,L-AP-4, -Conotoxin-GVIA (Fig. 3A,C) and nimodipine (Fig. 3A,B) further inhibited the Ba²⁺ current (by 10 and 20%, respectively), whereas -Agatoxin-IVA did not (Fig. 3B,D). Altogether these results demonstrated that mGluR7 selectively blocked the P/Q-type Ca²⁺ channels, without significantly affecting N-, L-, and R-types.

View larger version (27K): [in this window] [in a new window]   Figure 3.   Selective blockade of P/Q-type Ba2+ currents by D,L-AP-4. Absence of effect of D,L-AP-4 (500 µM) on -Agatoxin-IVA- (250 nM, A) and inhibitory effect of the agonist on -Conotoxin GVIA- (1 µM, B, D) and nimodipine- (1 µM, C, D) insensitive Ba2+ currents. Graphs A-D were obtained from four different mGluR7-transfected cerebellar granule cells. Similar results were obtained from at least five other cells, for each graph.

mGluR7-mediated activation of a G_o-like protein

To examine if a G-protein was involved in the mGluR7-mediated inhibition of P/Q-type Ba²⁺ currents, we intracellularly applied the nonselective G-protein activator, GTPS (100 µM). Under these conditions, the D,L-AP-4-mediated inhibition of Ba²⁺ current was highly reduced (Fig. 4A). Indeed, -Agatoxin-IVA inhibited only 12 ± 2% (n = 5) of the current, indicating that the P/Q-type Ca²⁺ channels were already significantly inhibited by GTPS. Overnight incubation of the culture in the presence of PTX (200 ng/ml) abolished the inhibitory effect of D,L-AP-4 (Fig. 4A). Together these observations showed that a G_i/o-like protein was involved in the D,L-AP-4-mediated inhibition of P/Q-type Ca²⁺ channels.

View larger version (27K): [in this window] [in a new window]   Figure 4.   D,L-AP-4 inhibited Ba2+ current through an indirect action of Go-protein. A, Mean (± SEM; n = 6-10) fractional reduction of the whole-cell Ba2+ current induced by D,L-AP-4 (500 µM) in mGluR7-transfected cerebellar granule cells, under different conditions (from left to right): control condition (CT), in the presence of intracellular GTPS (100 µM), after an overnight PTX treatment (200 ng/ml), after intracellular dialysis of an antibody raised against the G-protein o subunit (1:100 dilution; anti-oAb), after intracellular dialysis of the 1A I-II loop peptide (10 µM; I-II loop), in cells cotransfected with CD8-ARK chimera (ARK), after intracellular dialysis of purified G-protein subunits (50 µg/ml; ). NS, Not significantly different from control. B, Whole-cell Ba2+ currents evoked by depolarizing steps to +10 mV, from a holding potential of 80 mV, preceded (right) or not (left) by a prepulse to +80 mV. Note that D,L-AP-4 (500 µM) induced similar Ba2+ current inhibition in the presence or absence of prepulse depolarization. Similar results were observed in eight other mGluR7-transfected neurons.

A specific antibody raised against the G-protein o subunit, which did not recognize the G-protein i subunit (see Materials and Methods), was used to determine which type of G-protein was involved in the L-AP-4 effect. This antibody significantly inhibited the effect of D,L-AP-4 on the Ba²⁺ current (Fig. 4A) without significantly altering the current density in the absence of agonist (89 ± 7 pA/pF, n = 10 without antibody; 79 ± 8 pA/pF, n = 6, with antibody). The boiled antibody was without effect (36 ± 4%, D,L-AP-4-induced inhibtion of Ba²⁺ current, n = 5). These results showed that a G_o-, rather than a G_i-like protein, was involved in the D,L-AP-4-mediated effect.

We then investigated the involvement of the G-protein o and subunits. In heterologous expression systems, it has been shown that the P/Q-type Ca²⁺ channels can be blocked by a direct binding of G-protein subunits on the I-II loop of the 1A Ca²⁺ channel subunit (De Waard et al., 1997; Bourinet et al., 1999). To test the implication of the native G-protein subunits in the mGluR7-mediated inhibition of Ba²⁺ current in transfected cerebellar granule cells, these subunits were quenched in two ways. First, a peptide derived from the I-II loop of the 1A Ca²⁺ channel subunit (10 µM) was applied into the cell via the recording electrode. Second, the cDNA coding for a chimera composed of the CD8 membrane receptor antigen and of a domain containing the G-protein subunit binding site of ARK, was cotransfected with mGluR7 in the cerebellar neurons. Under both conditions, the inhibitory effect of D,L-AP-4 was strongly reduced (Fig. 4A). We verified that after dialysis of the I-II loop of the 1A or transfection of the CD8-ARK chimera, -Agatoxin IVA still inhibited the whole-cell Ba²⁺ current (35 ± 6% inhibition, n = 5, in dialyzed neurons; 37 ± 2% in transfected neurons), indicating that the P/Q-type Ca²⁺ channels were not significantly affected by the tested peptides. Also, perfusion of a peptide-free solution or transfection of the CD8-ARK chimera deleted of its binding site for G-protein subunits did not affect the action of D,L-AP-4 on Ba²⁺ currents (33 ± 3% inhibition, n = 5 in dialyzed neurons; 39 ± 3%, n = 5 in transfected neurons). These results indicated that mGluR7-mediated Ca²⁺ channel inhibition involved G-protein subunits.

However, dialysis of G-protein subunits (both at 50 µg/ml) did not significantly modify activation and inactivation kinetics of the whole-cell Ba²⁺ current (data not shown), and neither significantly altered the fraction of Ba²⁺ current that was inhibited by D,L-AP-4 (Fig. 4A), in mGluR7/GFP-cotransfected cerebellar granule cells. The tested G-protein subunits inhibited and slowed activation kinetics of P/Q-type Ca²⁺ channels expressed in Xenopus oocytes, indicating that the absence of effect of the tested G-protein subunits in cultured cerebellar granule cells did not result from a lack of activity of these molecules (data not shown). Together, these observations indicated that G-protein subunits were required, but not sufficient to mediate the mGluR7-induced inhibition of Ba²⁺ current.

We further examined whether the mGluR7-mediated Ba²⁺ current inhibition could result from the well characterized direct interaction of G_o-protein subunits with the I-II loop of 1A Ca²⁺ channel subunit (De Waard et al., 1997; Bourinet et al., 1999). A positive prepulse relieves this interaction and inhibition (voltage-dependent facilitation) of the P/Q-type Ba²⁺ current. In mGluR7-transfected cerebellar granule cells, no such voltage-dependent facilitation was observed in the absence or presence of D,L-AP-4 (Fig. 4B). Thus the ratio, R, between amplitudes of Ba²⁺ currents evoked with and without depolarizing prepulse was measured in the absence and presence of D,L-AP-4. We obtained values of R that were not significantly different from 1 (R = 1.10 ± 0.07%, n = 9, in the absence of agonist; R = 1.04 ± 0.06%, n = 9, in the presence of D,L-AP-4). This result indicated the absence of both tonic and mGluR7-mediated inhibition of Ca²⁺ channels through direct interaction of G_o-protein subunits in mGluR7-transfected cerebellar granule cells.

mGluR7-mediated PKC-dependent blockade of Ba²⁺ current

Because mGluR7 appeared to inhibit P/Q-type Ca²⁺ channels via an indirect action of a G_o-like protein, we searched for any involvement of additional intracellular factors. The protein kinase A inhibitors, Rp-cAMPS (10 µM) and PKI (1 µM), added to the recording pipette solution, failed to inhibit Ba²⁺ currents in the absence or presence of D,L-AP-4, in nontransfected as well as mGluR7-transfected cerebellar granule cells (data not shown). On the other hand, the PKC activator PDBu (1 µM) inhibited the whole-cell Ba²⁺ current of cultured cerebellar granule cells by 27 ± 5% (n = 7). Similar results were obtained with the other PKC agonist, PMA (200 nM; 21 ± 7% inhibition, n = 4). These effects were not additive to the inhibitory effect of -Agatoxin IVA (5 ± 3% inhibition after PDBu application, n = 7; Fig. 5A), indicating that P/Q-type Ca²⁺ channels in these neurons were PKC-sensitive. The hypothesis that mGluR7 activated a PKC-dependent pathway was therefore tested. In mGluR7-transfected granule neurons, a 30 min pretreatment with the selective PKC inhibitor GF109203X (10 µM) almost abolished the inhibitory effect of D,L-AP-4 (Fig. 5B). We then studied whether PLC was also involved. The D,L-AP-4-mediated inhibitory effect was altered by the PLC inhibitor U73122 (2 µM), whereas the inactive analog U73343 (same concentration) was without effect (Fig. 5B). These results indicated that mGluR7 blocked P/Q-type Ca²⁺ channels via a PLC/PKC-dependent pathway.

View larger version (19K): [in this window] [in a new window]   Figure 5.   D,L-AP-4-mediated inhibition of Ba2+ currents involved a PLC/PKC pathway in mGluR7-transfected cerebellar granule cells. A, Time course of the inhibitory effect of the PKC activator PDBu (1 µM, 30 min) on whole-cell Ba2+ currents and absence of effect of -Agatoxin-IVA (250 nM) after the PDBu-mediated inhibition. B, Mean (± SEM; n = 5-10) fractional reduction of the whole-cell Ba2+ current recorded in mGluR7-transfected cerebellar granule cells induced by D,L-AP-4 (500 µM), under the following conditions (from left to right): in control cells (CT), in cells pretreated for 30 min with the PKC antagonist GF109203X (10 µM), and after 5 min dialysis of the PLC antagonist U73122 (2 µM) or the inactive analog U73343 (same concentration).

Because inhibition of Ba²⁺ currents by transfected mGluR7 was PLC-dependent, we tested whether the native receptor was able to induce IP₃ formation in the studied neurons. In nontransfected cerebellar cultures, D,L-AP-4 at concentrations that stimulated mGluR7 (500 µM or 1 mM), increased IP₃ formation by more than twofold, whereas lower concentrations of D,L-AP-4 (100 µM and 200 µM) had no significant effect on basal IP₃ formation (Fig. 6A). The D,L-AP-4-induced formation of IP₃ was abolished by an overnight pretreatment of the cultures with PTX (200 ng/ml; Fig. 6A). Therefore, native mGluR7 in cerebellar granule cells induced formation of IP₃ via a G_o-protein-dependent pathway. This result was in agreement with our electrophysiological data. Similar increases of IP₃ formation were obtained in mGluR7 transfected cerebellar cultures (Fig. 6A). This apparent absence of effect of transfected mGluR7 can be explained by the low rate of transfection (2-5%) obtained with our method (Ango et al., 1999).

View larger version (58K): [in this window] [in a new window]   Figure 6.   D,L-AP-4-induced IP3 formation in nontransfected or mGluR7-transfected cultured cerebellar granule cells. A, IP formation was determined in nontransfected or mGluR7-transfected cerebellar cultures (from left to right) in the absence (Basal) and presence of the mGluR1 agonist DHPG (positive control), or different concentrations of D,L-AP-4. The last bar of the histogram on the right was obtained in PTX-treated cells. Each bar of the histogram represents the mean ± SEM of four independent experiments performed in triplicate. B, Mean (± SEM; n = 5-10) fractional reduction of the whole-cell Ba2+ current recorded in mGluR7-transfected cerebellar granule cells induced by D,L-AP-4 (500 µM), under the following conditions (from left to right): in control cells, in cells recorded with an intracellular medium containing 20 mM BAPTA, and after 5 min dialysis of the IP3 receptor antagonist heparin (400 µg/µl).

The observed mGluR7-induced IP₃ formation should lead to diacylglycerol synthesis and intracellular Ca²⁺ release from IP₃-sensitive stores. The released Ca²⁺, together with diacylglycerol, should then activate PKC, which in turn blocks P/Q-type Ca²⁺ channels. In agreement with this hypothesis, the inhibitory effect of D,L-AP-4 on Ba²⁺ currents was antagonized by the IP₃ receptor blocker heparin (400 µg/ml; Fig. 6B). Although resistant to 20 mM EGTA, the L-AP-4 induced P/Q type Ca²⁺ channel inhibition was abolished by the faster Ca²⁺ chelator BAPTA (20 mM), added to the intracellular recording medium (Fig. 6B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present results indicated that activation of mGluR7 selectively inhibited P/Q-type Ca²⁺ channels in cultured cerebellar granule cells. This blockade involved a G_o-protein and unexpectedly for a group III mGluR, PLC, intracellular Ca²⁺, and PKC activation. Consistent with these results, we found that mGluR7 stimulated neuronal IP₃ formation in a PTX-dependent manner. We therefore propose a model in which mGluR7 activates a G_o-protein, the subunits of which directly stimulated PLC, likely in combination with the o subunit, and induced IP₃ and diacylglycerol formation. This in turn results in intracellular Ca²⁺ release from IP₃-sensitive Ca²⁺ stores, PKC activation, and P/Q-type Ca²⁺ channel inhibition (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Figure 7.   Model for mGluR7-induced inhibition of the P/Q-type Ca2+ channels in cerebellar granule cells. mGluR7 activates a Go protein, the o and subunits of which stimulates a PLC. This results in IP3 and diacylglycerol (DAG) formation. IP3-induced Ca2+ release and DAG stimulate PKC, which in turn blocks the P/Q-type Ca2+ channel. Whether PKC directly phosphorylates the channel or acts on an intermediate protein is not determined.

Transfected granule cells as a model to study the mGluR7 signaling

The cultured cerebellar granule cell preparation provided a more physiological environment than the classical heterologous expression system to study the transduction signaling of transfected neuronal receptors. At the concentrations presently used (0.5 and 1 mM), D,L-AP-4 could not distinguish between the different group III mGluRs. Because the agonist did not affect Ba²⁺ currents in the absence of transfected mGluR7, native functional group III mGluRs were likely absent at the somatic plasma membrane of cultured cerebellar granule cells. This was confirmed, at least for mGluR7, by our immunolabeling experiments. Indeed, the native receptor was strictly localized in neuritic processes, whereas the transfected receptor was also detected at the somatic membrane. Together, these observations indicated that the D,L-AP-4 effects that we observed on Ba²⁺ currents in mGluR7-transfected cells certainly resulted from selective activation of this receptor, with the exclusion of any other group III mGluRs.

It could be argued that the unexpected coupling of the transfected mGluR7 with PLC pathway resulted from overexpression of the receptor in the cell body. Although not definitively proved, the following results argued against this hypothesis. First, the native neuritic mGluR7 also activated PLC, as indicated by the D,L-AP-4-mediated IP₃ formation observed in nontransfected cerebellar cultures. Second, cotransfection and overexpression of mGluR7 with mGluR2 did not change the coupling characteristics of the latter receptor (our unpublished observations), i.e., selective inhibition of N- and L-type Ca²⁺ channels (Chavis et al., 1995, 1998).

Indirect G_o-protein-mediated inhibition of P/Q-type Ca²⁺ channels

The mechanisms by which mGluRs block Ca²⁺ channels in neurons remain controversial. On one hand, a membrane delimited action mediated by direct interaction between G_o-protein subunits and Ca²⁺ channels has been proposed (Trombley and Westbrook, 1992; Choi and Lovinger, 1996; Ikeda, 1996). On the other hand, a slow inhibition of Ca²⁺ channels recorded in cell-attached patches has also been found and was consistent with the involvement of a soluble intracellular messenger (Chavis et al., 1994). The nature of this messenger remains however to be determined. In the present study, inhibition of P/Q-type Ca²⁺ channels involved a G_o-like protein, the subunits of which did not seem to directly interact with the Ca²⁺ channel, because this inhibition was neither accompanied by slow activation kinetics of the Ba²⁺ current, nor removed by a depolarizing prepulses. A direct voltage-insensitive action of G-protein o subunit on Ca²⁺ channels has been reported in sympathetic neurons (Delmas et al., 1998). However, such a mechanism did not seem to be involved in cerebellar granule cells because inhibition of PLC or PKC completely abolished the effect of D,L-AP-4 on I_Ca (Fig. 5B).

Now, cloned P/Q-types Ca²⁺ channels are generally inhibited by direct interaction of G-protein subunits with the I-II loop of the 1A Ca²⁺ channel subunit (De Waard et al., 1997). However, it has been recently reported that alternative splicing of the 1A gene generates 1A-a and 1A-b subunits with distinct properties. Thus, the presence of a Val₄₂₁ residue in the I-II linker domain of the 1A-b (absent in the 1A-a) splice variant subunit confers to the Ca²⁺ channel the faculty of being directly inhibited by G-protein subunits (Bourinet et al., 1999). The authors' data also predict that most of 1A transcript in cerebellar neurons should be of the 1A-a subtype. Together these data are consistent with the hypothesis that cultured cerebellar granule cells may predominantly express the 1A-a splice variant subunit, which could explain the absence of direct effect of G_o-protein subunits on P/Q-type Ca²⁺ currents in these neurons on application of mGluR7 agonist.

Activation of G_o-protein generally leads to inhibition of N-type Ca²⁺ channels (Hille, 1994) through a direct action of the G-protein subunits on the channels (Ikeda, 1996), whereas in the present study N-type Ca²⁺ channels were spared. It is worth noting that in cultured cerebellar granule cells, inhibition of N-type Ca²⁺ channels was mediated by the G_o-protein-coupled mGluR2/3, and this effect did not display the fast kinetics and membrane-delimited voltage-dependent characteristics of a direct action of G_o-protein subunits on these channels (Chavis et al., 1994, 1995). This suggests that other mechanisms are involved in our preparation and mediate a selective inhibition of P/Q-type versus N-type Ca²⁺ channels by mGluR7 and mGluR2/3, respectively. Two tentative hypotheses can be proposed to explain such a selectivity. mGluRs may be colocalized with specific Ca²⁺ channels in functional microdomains, probably through interaction with scaffold proteins, the nature of which remains however to be identified. Alternatively, particular G-protein subunit combinations may activate specific signaling pathways. For instance, it has been reported that distinct o, , and  subunit combinations display different efficacy in modulating ARK (Muller et al., 1993) or voltage-gated Ca²⁺ channel activity (Kleuss et al., 1993; Kalkbrenner et al., 1995).

Cellular determinants of the GluR7-mediated activation of PLC

Unexpectedly, we found that the mGluR7-mediated inhibition of the Ca²⁺ channels was PLC-dependent. The effect was abolished by treatments with PTX or a specific antibody raised against the G-protein o subunit, indicating that a G_o protein activated PLC in cultured cerebellar granule cells. This finding was reminiscent of the G_o-mediated activation of PKC in enteric neurons (Pan et al., 1997). The effect of mGluR7 was mimicked by the nonselective G-protein activator GTPS and blocked by quenching the G-protein subunits with intracellular specific peptides. A likely hypothesis is that G-protein subunits directly acted on a PLC in our preparation, as it is the case in various heterologous expression systems (Blank et al., 1992; Boyer et al., 1992, 1994; Camps et al., 1992; Blitzer et al., 1993). However, it is worth noting that intracellular dialysis of purified G-protein subunits did not mimic the inhibitory effect of mGluR7 on Ba²⁺ currents. Together these observations indicated that G-protein subunits were required, but not sufficient to activate PLC in neurons, and that G-protein o and subunits were both involved. Although G-protein o subunit reconstituted in phospholipid vesicles was not required to activate PLC, this subunit shifted to the left the concentration-effect curve for -mediated activation of PLC and increased the maximal activity of PLC (Boyer et al., 1992). It is therefore possible that under our experimental conditions, such a synergistic effect of G-protein o subunits on activation of a PLC by the subunits was required to reach a level of PLC activity sufficient to activate PKC and inhibit P/Q-type Ca²⁺ channels.

It has been shown that PKC phosphorylates a site located in the domain I-II linker of the cloned 1A Ca²⁺ channel subunit, which results in upregulation of the P/Q-type Ca²⁺ current (Bourinet et al., 1999). Because phorbol esters or mGluR7 inhibited native P/Q-type Ca²⁺ channels in cultured cerebellar granule cells, a different PKC phosphorylation site was involved in this effect. An alternative hypothesis is that PKC phosphorylated an intermediate protein that in turn downregulated the Ca²⁺ channel.

A reason why native P/Q-type Ca²⁺ channels in cultured cerebellar granule cells were not sensitive to the facilitatory effect of PKC could be that these neurons express the 1A-a Ca²⁺ channel subunit isoform, as suggested above. Indeed, in addition to be little sensitive to G-protein subunits interaction, this subunit isoform harbors low sensitivity to upregulation mediated by PKC (Bourinet et al., 1999).

Possible physiological consequences of the mGluR7-mediated inhibition of P/Q-type Ca²⁺ channels

Activation of PLC by mGluR7 was sufficient to inhibit Ca²⁺ channels in cultured cerebellar granule cells. This observation was consistent with the absence of effect of PKA inhibitors on Ba²⁺ currents, in the absence or presence of D,L-AP-4, in mGluR7-transfected cerebellar granule cells. Moreover, it has been reported that L-AP-4 activates cAMP-independent and PLC-dependent pathways in mitral olfactory bulb (Schoppa and Westbrook, 1997) and retinal ganglion cells (Shen and Slaughter, 1998) respectively. Also, the efficiency of the coupling between mGluR7 and adenylyl cyclase in BHK cells is relatively low (Saugstad et al., 1994). Altogether these results suggest that the classical inhibition of adenylyl cyclase by group III mGuRs, which has been described in heterologous expression systems (Okamoto et al., 1994; Saugstad et al., 1994; Wu et al., 1998), would not be the primary transduction pathway by which mGluR7 triggers its physiological effects in neurons. We therefore propose that in natural systems this receptor, like group I mGluRs, acts through a PLC-dependent cascade.

To further understand the role of mGluR7 in synaptic transmission, one needs to transpose our results to neuronal synaptic terminals. Like mGluR7 (Shigemoto et al., 1996, 1997; Kinzie et al., 1997), IP₃-sensitive Ca²⁺ stores, PKC activity (Rodriguez-Moreno et al., 1998), and P/Q-type Ca²⁺ channels (Turner et al., 1992; Takahashi and Momiyama, 1993; Regehr and Mintz, 1994; Dunlap et al., 1995) have been found at presynaptic sites and control neurotransmitter release. Therefore, our results anticipate that mGluR7 would downregulate synaptic transmission through activation of PKC. This hypothesis is in apparent discrepancy with the classical phorbol ester-mediated facilitation of transmitter release, but it has been shown that these compounds act independently of PKC, the major brain isoform of PKC (Goda et al., 1996).

This does not exclude the possibility that other presynaptic receptors can mediate direct effects of G_o-protein on N-type Ca²⁺ channels. Indeed, it has been shown that although N-type Ca²⁺ channels can be inhibited by direct and indirect effects of G-proteins in sympathetic neuron somata, only the direct pathway seems to mediate inhibition of transmitter release (Koh and Hille, 1997). The relative importance of a direct versus indirect inhibition of presynaptic Ca²⁺ channels may depend on the colocalization of Ca²⁺ channels with G_o-protein-coupled receptors. Thus, although the model we propose here (Fig. 7) provides a possible physiological mechanism by which mGluR7 could dampen synaptic transmission, it may not be predominant under low-frequency synaptic activity. Indeed, whereas neurotransmitter glutamate normally lasts shortly in the synaptic cleft, the mGluR7-induced inhibition of Ba²⁺ current needed a long time to develop and lasted long after washout. Because of these kinetic properties, the mGluR7-activated pathway may be more important under sustained synaptic activity such as during induction of synaptic plasticity or subthreshold epileptic neuronal discharges. In agreement with this hypothesis, inhibition of excitatory synaptic transmission by group III mGluRs, during high- but not low-frequency synaptic activity, has been observed in locus coeruleus (Dube and Marshall, 2000). This hypothesis provides the mGluR7 pathway as neuroprotective and is consistent with physiological studies showing that adult mGluR7 knock-out mice died from epileptic seizures (Masugi et al., 1999).

	FOOTNOTES

Received April 3, 2000; revised July 20, 2000; accepted Aug. 16, 2000.

This work was supported by Centre National de la Recherche Scientifique and grants from Association Française contre les Myopathies, Fondation pour la Recherche Médicale, Bayer (France), and Hoechst-Marrion-Roussel (FRHMR1/9702). We thank J. P. Pin and F. Ango for constructive discussion of this work. We also thank Dr. J. Saugstad (Atlanta, GA) for the rat mGluR7a cDNA, J. M. Sabatier (Marseille, France) for the synthesis of the 68 AA peptide, V. Homburger (Montpellier, France) for the anti-Go antibody, and B. Mouillac (Montpellier, France) for the anti-cMyc monoclonal antibody.

Correspondence should be addressed to Dr. L. Fagni, Centre National de la Recherche Scientifique, Unité Propre Recherche 9023, CNRS-INSERM de Pharmacologie et d'Endocrinologie, 141 Rue de la Cardonille, 34000 Montpellier, France. E-mail: fagni{at}bacchus.montp.inserm.fr.

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