FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019934881> ?p ?o ?g. }

• W2019934881 endingPage "1228" @default.
• W2019934881 startingPage "1223" @default.
• W2019934881 abstract "G protein-coupled receptors mobilize neuronal signaling cascades which until now have not been shown to depend on the state of membrane depolarization. Thus we have previously shown that the metabotropic glutamate receptor type 7 (mGlu7 receptor) blocks P/Q-type Ca2+ channels via activation of a Go protein and PKC, in cerebellar granule cells. We show here that the transient depolarizations used to evoke the studied Ca2+ current were indeed permissive to activate this pathway by a mGlu7 receptor agonist. Indeed, sustained depolarization to 0 mV was sufficient to inhibit P/Q-type Ca2+ channels. This effect involved a conformational change in voltage-gated sodium channel independently of Na+ flux, activation of a pertussis toxin-sensitive G-protein, inositol trisphosphate formation, intracellular Ca2+ release, and PKC activity. Subliminal sustained membrane depolarization became efficient in inducing inositol trisphosphate formation, release of intracellular Ca2+ and in blocking Ca2+ channels, when applied concomitantly with the mGlu7a receptor agonist,d,l-aminophosphonobutyrate. This synergistic effect of membrane depolarization and mGlu7 receptor activation provides a mechanism by which neuronal excitation could control action of the mGlu7 receptor in neurons. G protein-coupled receptors mobilize neuronal signaling cascades which until now have not been shown to depend on the state of membrane depolarization. Thus we have previously shown that the metabotropic glutamate receptor type 7 (mGlu7 receptor) blocks P/Q-type Ca2+ channels via activation of a Go protein and PKC, in cerebellar granule cells. We show here that the transient depolarizations used to evoke the studied Ca2+ current were indeed permissive to activate this pathway by a mGlu7 receptor agonist. Indeed, sustained depolarization to 0 mV was sufficient to inhibit P/Q-type Ca2+ channels. This effect involved a conformational change in voltage-gated sodium channel independently of Na+ flux, activation of a pertussis toxin-sensitive G-protein, inositol trisphosphate formation, intracellular Ca2+ release, and PKC activity. Subliminal sustained membrane depolarization became efficient in inducing inositol trisphosphate formation, release of intracellular Ca2+ and in blocking Ca2+ channels, when applied concomitantly with the mGlu7a receptor agonist,d,l-aminophosphonobutyrate. This synergistic effect of membrane depolarization and mGlu7 receptor activation provides a mechanism by which neuronal excitation could control action of the mGlu7 receptor in neurons. inositol trisphosphate green fluorescent protein intracellular [Ca2+] 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrodotoxin pertussis toxin voltage-gated sodium channel The excitatory neurotransmitter, glutamate, mediates its effects by activating ionotropic and metabotropic (mGlu) receptors. Eight genes encoding mGlu receptors have been identified and classified into three groups. The group I (mGlu1 and mGlu5 receptors) activates PLC through a Gq protein, whereas group II (mGlu2 and mGlu3 receptors) and group III (mGlu4, mGlu6, mGlu7, and mGlu8 receptors) are coupled to Gi/Go proteins (1Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Google Scholar). These receptors are widely distributed throughout the mammalian brain (2Kinzie J.M. Saugstad J.A. Westbrook G.L. Segerson T.P. Neuroscience. 1995; 69: 167-176Google Scholar, 3Ohishi H. Akazawa C. Shigemoto R. Nakanishi S. Mizuno N. J. Comp. Neurol. 1995; 360: 555-570Google Scholar, 4Bradley S.R. Levey A.I. Hersch S.M. Conn P.J. J. Neurosci. 1996; 16: 2044-2056Google Scholar, 5Kinoshita A. Shigemoto R. Ohishi H. van der Putten H. Mizuno N. J. Comp. Neurol. 1998; 393: 332-352Google Scholar), but only the mGlu7 receptor subtype is almost exclusively localized at presynaptic sites (6Kinzie J.M. Shinohara M.M. van den Pol A.N. Westbrook G.L. Segerson T.P. J. Comp. Neurol. 1997; 385: 372-384Google Scholar, 7Shigemoto R. Kulik A. Roberts J.D. Ohishi H. Nusser Z. Kaneko T. Somogyi P. Nature. 1996; 381: 523-525Google Scholar, 8Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J. Neurosci. 1997; 17: 7503-7522Google Scholar). Group II and III mGlu receptors, and particularly the mGlu7 receptor subtype, inhibit synaptic transmission (1Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Google Scholar, 9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Google Scholar). Thus in vitrostudies have shown that stimulation of mGlu7 receptors decreases release of glutamate in cerebellar cultures (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar) and GABA in striatal cultures (11Lafon-Cazal M. Viennois G. Kuhn R. Malitschek B. Pin J.P. Shigemoto R. Bockaert J. Neuropharmacology. 1999; 38: 1631-1640Google Scholar), promoting neuroprotection and excitotoxicity, respectively. Moreover, we have recently shown that mGlu7 receptors selectively block P/Q-type Ca2+ channels in neurons (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar), and these channels control transmitter release (13Dunlap K. Luebke J.I. Turner T.J. Trends Neurosci. 1995; 18: 89-98Google Scholar). Together these studies suggest that mGlu7 receptors play an important role in the modulation of synaptic transmission. Recent studies have pointed out that in the rat locus coeruleus, group III mGlu receptor agonists down-regulate high but not low frequency synaptic activity (14Dube G.R. Marshall K.C. J. Neurophysiol. 2000; 83: 1141-1149Google Scholar), suggesting that this receptor action depends on the state of neuronal depolarization. Moreover, a voltage-dependent activation of a Go protein has recently been shown in rat brain synaptoneurosomes (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar). In light of these results, and because the mGlu7 receptor is coupled to a Go protein, this receptor is a potential candidate for mobilization of both voltage- and Goprotein-dependent events in neurons. We therefore investigated, in cultured cerebellar granule cells, the effect of membrane depolarization on this receptor signaling. We have previously shown that the mGlu7 receptor-induced blockade of P/Q-type Ca2+ channels is independent of a direct action of Go protein βγ subunits on the Ca2+channels, but results from IP31 formation and PKC activation (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar). Here we show a synergistic effect of the receptor activation and membrane depolarization. Primary cultures of mouse cerebellar granule cells were prepared as previously described (16Van Vliet B.J. Sebben M. Dumuis A. Gabrion J. Bockaert J. Pin J.P. J. Neurochem. 1989; 52: 1229-1239Google Scholar). Since these cultured neurons do not express functional native group III mGlu receptors in the soma (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar), they were transfected with an expression plasmid containing the mGlu7a receptor protein, using a method described elsewhere (17Ango F. Albani-Torregrossa S. Joly C. Robbe D. Michel J.M. Pin J.P. Bockaert J. Fagni L. Neuropharmacology. 1999; 38: 793-803Google Scholar). As only 10–20% neurons were transfected using this method, the mGlu7a receptor was co-transfected with the transfection marker, green fluorescent protein (GFP), for single cell electrophysiological recording and intracellular Ca2+ concentration ([Ca2+]i) measurement. Barium currents were recorded using the whole cell configuration of the patch clamp technique, at room temperature, from mGu7a/GFP-expressing cerebellar granule cells, after 9 ± 1 days in vitro. The bathing medium contained (mm): BaCl2 (20Blahos J. Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Google Scholar), HEPES (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), tetraethylammonium acetate (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), glucose (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), and Na acetate (120), adjusted to pH 7.4 with Na-OH and 330 mOsm with Na acetate. Drug solutions were prepared in this medium and pH of the solutions was readjusted to 7.4 with NaOH. The NMDA receptor-channel blocker, MK-801 (1 μm), was added to all the solutions in order to avoid activation of this receptor by the d-isoform ofd,l-AP4. 2L. Fagni and M. Lafon-Cazal, unpublished observation. We also added TTX (0.3 μm), 6-Cyano-7-nitroquinoxaline-2,3-dione (100 μm), and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt 250 μm), in order to block Na+ flux through VGSCs and release of glutamate, as well as activation of ionotropic glutamate receptors and endogenous mGlu1 receptors. Indeed, cultured cerebellar granule neurons do not express functional native mGlu5 receptors (17Ango F. Albani-Torregrossa S. Joly C. Robbe D. Michel J.M. Pin J.P. Bockaert J. Fagni L. Neuropharmacology. 1999; 38: 793-803Google Scholar, 18Ango F. Pin J.P. Tu J.C. Xiao B. Worley P.F. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 8710-8716Google Scholar, 19Prezeau L. Carrette J. Helpap B. Curry K. Pin J.P. Bockaert J. Mol. Pharmacol. 1994; 45: 570-577Google Scholar). Patch pipettes had resistances of 3–5 mΩ when filled with the following internal solution (mm): Cs-acetate (100), CsCl (20Blahos J. Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Google Scholar), MgCl2 (2Kinzie J.M. Saugstad J.A. Westbrook G.L. Segerson T.P. Neuroscience. 1995; 69: 167-176Google Scholar), HEPES (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), glucose (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar), EGTA (20 mm), Na2ATP (2 mm), and cAMP (1 mm), adjusted to pH 7.2 with CsOH and 300 mOsm with Cs-acetate. In some experiments, intracellular EGTA was replaced by BAPTA. Recordings started at least 5 min after breaking the membrane patch and stabilization of evoked Ba2+ current (IBa). Barium currents were evoked using voltage-clamp pulses of 500 ms duration, from a holding potential of −80 mV to a test potential of 0 mV, at a rate of 0.1 Hz, except when specified in the text. Membrane resistance was measured by applying hyperpolarizing pulses of 20 mV amplitude and 55 ms duration, from a holding potential of −80 mV. Current signals were recorded using an Axopatch 200 amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz with an 8-pole Bessel filter and sampled at 3 kHz on a Pentium II PC computer. Analyses were performed using the pClamp6 program of Axon Instruments. Barium currents were measured at their peak amplitude and expressed as mean ± S.E. of the indicated number (n) of experiments. The procedure to measure IP accumulation in neurons was adapted from a one previously described (20Blahos J. Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Google Scholar). Briefly, 1-week-old cerebellar cultures were incubated for 14 h in culture medium containing 2 μCi/mlmyo-[3H]inositol (23.4 Ci/mol; Invitrogen, Paris, France). Cells were then washed 3 times and incubated for 1 h at 37 °C in 1 ml of HEPES saline buffer (146 mm NaCl, 4.2 mm KCl, 0.5 mm MgCl2, 0.1% glucose, 20 mm HEPES, 0.3 μm TTX, 10 μm MK801, 100 μm6,7-Dinitroquinoxaline-2,3-dione, and 250 μmCPCCOEt, pH 7.4) supplemented with 1 unit/ml glutamate pyruvate transaminase (Roche Molecular Biochemicals, Meylan, France) and 2 mm pyruvate (Sigma, St. Quentin-Fallavier, France). In experiments where KCl was elevated to 30 or 100 mm, the same respective amounts of NaCl were removed from the saline buffer solution. This induced theoretical membrane steady-state depolarizations to −40 and −10 mV, respectively, at 20 °C, assuming an intracellular K+ concentration of 140 mm. Cells were then washed again with the same saline 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 IP purified on Dowex columns. Total radioactivity remaining in the membrane fraction was counted after treatment with 10% Triton X-100, 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. Similar results were obtained in mGlu7 receptor-transfected and nontransfected cultures. Note that only data obtained from transfected cultures are presented here. Given that only 10–20% neurons were transfected with our method, this indicated that majority ofd,l-AP4-induced IP accumulation measured in mGlu7 receptor-transfected cultures resulted from activation of the native mGlu7 receptors (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar). Intracellular Ca2+ level was measured in single cells, as previously described (21Boccara G. Choby C. Frapier J.M. Nargeaot J. Dayanithi G. Richard S. Circ. Res. 1999; 85: 606-613Google Scholar). Briefly, the dual excitation ratiometric Ca2+-sensitive dye, Fura-2, was used for [Ca2+]i imaging measurements by means of an Olympus-LSR system (MERLIN; Life Science Resources Ltd., Cambridge, UK). Cerebellar cultures were loaded by incubation with 2.5 μm Fura-2-acetoxymethyl ester and 0.02% pluronic acid (F-127; Molecular Probes Inc., Eugene, OR) for 35 min at 37 °C in a buffer medium containing (mm): NaCl (150), KCl (3Ohishi H. Akazawa C. Shigemoto R. Nakanishi S. Mizuno N. J. Comp. Neurol. 1995; 360: 555-570Google Scholar), MgCl2 (4Bradley S.R. Levey A.I. Hersch S.M. Conn P.J. J. Neurosci. 1996; 16: 2044-2056Google Scholar), glucose (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), HEPES (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar), TTX (3 × 10−4), MK801 (10−2), 6,7-Dinitroquinoxaline-2,3-dione (10−1), and CPCCOEt (25 × 10−2), pH 7.4, adjusted with NaOH. This medium was used to thoroughly rinse the cells before mounting them on the stage of the inverted microscope of the Olympus-LSR system and to prepare drug solutions. In experiments where 30 mm KCl was added to the medium, NaCl concentration was lowered to 120 mm. The osmolarity of all the solutions was adjusted to 330 mOsm with NaCl. Epifluorescent illumination was via a SpectraMASTER monochromator (Life Science Resources Ltd.) coupled to the microscope fitted with a UV transparent oil objective (Uapo/340 40X/1.35). The image was detected with an Astrocam 12/14 bit frame transfer digital camera (Life Science Resources Ltd.). The Olympus-LSR system controlled the illuminator and camera, and performed image ratioing and analysis. The intensity of fluorescent light emission at λ = 510 nm, using excitation at 340 and 380 nm, was monitored for each single cell analyzed. The ratio of fluorescence emission when excited at 340 nm (the absorbance peak of Fura-2 bound to Ca2+) to 380 nm (the absorbance peak of free Fura-2) was used as an index of [Ca2+]i, with an increase in the fluorescence ratio (F340/F380) signifying an increase in [Ca2+]i. Although [Ca2+]i measurements were performed in cultured cerebellar granule cells expressing the GFP reporter gene, one can estimate that less than 1% of the signal at 340 nm excitation and less than 5% of the signal at 380 nm excitation were contributed by GFP. d,l-AP4, 6-Cyano-7-nitroquinoxaline-2,3-dione, and MK-801 were purchased from Tocris Cockson (Bristol, UK), PTX, nimodipine, GF109203X, and veratridine from RBI (Sigma), ω−agatoxin-IVA from Alomone Labs (Jerusalem, Israel), heparin from Calbiochem (Meudon, France), TTX from Latoxan (Valence, France), Fura-2 from Molecular Probes (Eugene, OR), and the GFP (pEGFP-N1) expression plasmid fromCLONTECH (Ozyme, Montigny-le-Bretonneux, France). The R- and S-enantiomers of DPI 201–106 were generous gifts from G. Romey (CNRS UPR 411, Nice, France). Stimulation of mGlu7 receptors byd,l-AP4 (500 μm) had no effect on somatic whole cell IBa, in nontransfected cerebellar granule cells, which was consistent with the presynaptic location of the receptor in neurons (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar). Therefore experiments were performed in cultured cerebellar granule cells transfected with a mGlu7 receptor expression plasmid. The receptor was expressed in both cell body and neurites which allowed us to study its effect on somatic IBa. In mGlu7 receptor-transfected cerebellar granule cells application of the receptor agonist, d,l-AP4 (500 μm), at a steady state potential of −80 mV, did not affect IBa evoked at the end of the application (mean ± S.E. = 1 ± 1% inhibition; n = 6; Fig.1, period 1). In contrast, application of d,l-AP4, combined with transient and repetitive depolarizations (which evoked IBa), induced a progressive and potent inhibition (39 ± 2%; n = 6) of the current that last upon wash out of the agonist (Fig. 1,period 2) (12Perroy J. De Prézeau L. Waard M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Google Scholar). These results indicated that mGlu7 receptors inhibited Ca2+ channels under conditions where cerebellar granule cells were transiently and repetitively depolarized, but not in resting neurons. Because inhibition of Ca2+ channels by mGlu7 receptors was dependent on transient and repetitive depolarization, we examined the effect of membrane depolarization per se (in the absence of receptor agonist) on the channel activity. To mimic physiological activity, we stimulated neurons using 20-ms depolarization pulses, from −80 to 0 mV. When applied at a frequency of 0.1 Hz, these stimulations evoked IBa of stable amplitude over a period of at least 45 min (Fig.2A, open circles). Increasing the frequency of the depolarization pulses up to 25 Hz decreased IBa amplitude (Fig. 2A, points). This frequency-dependent decrease in amplitude of the current was reversible immediately after cessation of the tetanic stimulation if the train of depolarization pulses last for less than 30 s (Fig. 2A, 1). On the other hand, a 1-min train of transient depolarizations induced a long lasting inhibition of the current (Fig. 2A, 2). To quantify this blockade we replaced the high frequency train of stimulation by a single square pulse depolarization of equivalent duration. Thus maintaining the cell for 1 min at a steady state potential of −80 mV did not alter IBa evoked after this resting period (0 ± 1% inhibition,n = 6, Fig. 2B, 1). On the other hand, holding the cell for 1 min at 0 mV inhibited IBa evoked after cessation of this steady state depolarization period (40 ± 2% inhibition;n = 6; Fig. 2B, 2). This effect was voltage- (Fig. 2C) and time-dependent (Fig.2D). After the sustained depolarization period, neither membrane resistance (not shown), nor IBa inactivation kinetics (Fig.2E) were significantly altered, indicating that inhibition of IBa did not result from change in passive properties of the membrane or biophysical properties of the Ca2+ channels. We interpreted the reversible decrease in IBa amplitude observed after a short train of stimulations or sustained depolarization shorter than 10 s, as a classical Ca2+ channel inactivation. On the other hand, we tentatively interpreted the long lasting decrease induced by longer depolarization trains, or sustained steady state depolarization, as a pure Ca2+ channel inhibition. The following data supported this hypothesis. First, IBa evoked after a sustained depolarization to 0 mV (Fig. 2B, 2), although of smaller amplitude, still displayed similar inactivation kinetics (Fig. 2E). Second, IBa inhibition was mediated through intracellular messengers. Thus, inhibition of IBa induced by sustained depolarization was abolished by PTX, the PKC inhibitor GF109203X, intracellular dialysis of the IP3 receptor antagonist heparin, or intracellular Ca2+ chelator BAPTA (Fig. 3A). Moreover, KCl-induced depolarization increased basal IP accumulation, in a PTX-dependent manner (Fig. 3B). Taken together these results suggested that inhibition of Ca2+ channels induced by sustained depolarization was distinct from their classical voltage-dependent inactivation, since it involved a Gi/Go protein, intracellular Ca2+, and PKC. We then searched for the voltage sensor of this inhibitory pathway. Since voltage-induced inhibition of IBa was blocked by PTX (Fig.3A), the voltage-sensitive step of this phenomenon should be either upstream Gi/Go protein activation, or the Gi/Go protein itself. Previous studies have shown the existence of a Go protein activation through a conformational change of VGSCs, regardless of the Na+current (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar). Since cerebellar granule cells express functional somatic VGSCs (−145 pA/pF Na+ current) (17Ango F. Albani-Torregrossa S. Joly C. Robbe D. Michel J.M. Pin J.P. Bockaert J. Fagni L. Neuropharmacology. 1999; 38: 793-803Google Scholar), we examined whether these channels were involved in the depolarization-induced inhibition of Ca2+ channels. Since experiments were performed in the presence of TTX, the effects observed here were obviously independent of Na+ flux through VGSCs. TheR-4-[3-(4-diphenylmethyl1-piperazinyl)-2-hydroxypropoxyl]-1H-indole-2-carbonitrile (R-DPI 201–106; 50 μm, 10 min), a drug that blocks VGSC conformational changes (22Romey G. Quast U. Pauron D. Frelin C. Renaud J.F. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 896-900Google Scholar), also blocked the voltage-induced inhibition of IBa (2 ± 5% inhibition, n = 8, Fig. 4A). Interestingly, this drug has been shown to block depolarization induced activation of Go protein in synaptoneurosomes (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar). The potent and selective VGSC activator, veratridine, shifted the voltage-induced inhibition of IBa toward more negative potentials (Fig. 6B,filled triangles). The S-DPI 201–106 (50 μm, 10 min treatment), which mimics the action of veratridine on VGSC (22Romey G. Quast U. Pauron D. Frelin C. Renaud J.F. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 896-900Google Scholar), also mimicked the effect of veratridine on IBa inhibition (data not shown). In the absence of sustained depolarization, neither veratridine nor S- or R-DPI 201–106 significantly altered IBa (1 ± 2, 2 ± 4, and 2 ± 1% inhibition, respectively; n = 10 for both conditions). These results suggested that VGSCs were the voltage sensors that triggered the depolarization-induced inhibition of Ca2+ channels.Figure 6Synergistic action of membrane depolarization or veratridine and d,l-AP4.A, IBa amplitude was expressed as described in the legend to Fig. 1. A 1-min steady-state depolarization to −40 mV was applied in the absence or presence of d,l-AP4. Note that only concomitant depolarization and d,l-AP4 application inhibited IBa. B, percentage inhibition of IBa expressed as a function of membrane potential (1 min steady-state depolarization), obtained under the indicated conditions. Each value is the mean ± S.E. of at least five experiments. C, inositol phosphate formation measured under the indicated conditions. *, significantly different from basal (p < 0.01).D, intracellular Ca2+ release induced byd,l-AP4- and/or 30 mm KCl (average of 15 individual neurons).View Large Image Figure ViewerDownload (PPT) We then explored which type of Ca2+ channel was inhibited by the sustained depolarization. After application of ω-agatoxin-IVA (250 nm), the toxin-resistant IBa was not significantly affected by subsequent sustained depolarization (38 ± 2% inhibition with ω-agatoxin-IVA; 2 ± 0.4% inhibition with depolarization, n = 7, Fig. 4B) and vice versa (40 ± 1.8% inhibition with depolarization; 2. 5 ± 1.2% inhibition with subsequent ω-agatoxin-IVA application,n = 8, Fig. 4C). The Ca2+channels inhibited by sustained depolarization were therefore of the P/Q-type. Since the mGlu7 receptor and membrane depolarization shared common mechanisms, inhibition of IBa by these factors should be mutually occluded. This hypothesis was confirmed by the following experiments. We have seen here above (Fig. 1) that in mGlu7 receptor-transfected cerebellar granule cells, application of the receptor agonist,d,l-AP4 (500 μm), during repetitive and transient depolarizations, progressively inhibited IBa (39 ± 2% inhibition; n = 6). Interestingly, the amount of IBa remaining after this action ofd,l-AP4 was not significantly affected by subsequent sustained depolarization (36 ± 3% inhibition withd,l-AP4; 3 ± 2% inhibition with depolarization; n = 5; Fig.5A). Conversely, after sustained depolarization, d,l-AP4 application (associated with repetitive transient depolarizations) did not further inhibit the remaining IBa (39 ± 3% inhibition with depolarization; 1 ± 1% inhibition withd,l-AP4; n = 6; Fig.5B). These results indicated a mutual occlusion of the effects of sustained depolarization and mGlu7 receptor activation. We then assessed whether or not voltage and mGlu7 receptor could act synergistically to inhibit Ca2+ channels. Transient and successive depolarization pulses applied at 0.1 Hz frequency (Fig. 1), or a 1-min steady-state depolarization to −40 mV (Fig.6, A, 1, andB, filled circles), did not per seaffect IBa. Similarly to these subliminal stimulations,d,l-AP4 applied at a steady state potential of −80 mV did not alter IBa (Figs. 1 and 6, B, open circles). However, a 1-min steady-state depolarization to −40 mV, combined with a d,l-AP4 application inhibited IBa by 20% (Fig. 6, A, 2, andB, open circles). These results indicated a synergistic inhibitory effect of mGlu7 receptors and membrane depolarization on IBa. We verified that this synergistic effect involved VGSCs. Application of the VGSC blocker, R-DPI 201–106, antagonized the action of d,l-AP4 applied concomitantly with transient depolarization pulses (7 ± 3% inhibition; n = 5). Moreover, co-application, but not separate applications, of veratridine andd,l-AP4, at a membrane potential of −60 mV, significantly inhibited IBa (Fig. 6B, filled andopen triangles). These results indicated that the mGlu7 receptor action depended on activation of VGSCs. This synergistic effect of mGlu7 receptor and membrane depolarization was also observed on the receptor intracellular signaling cascade. Thus, basal IP level was not affected by 30 mm KCl (equivalent to −40 mV depolarization), nor byd,l-AP4 alone, but increased when KCl andd,l-AP4 were co-applied (Fig. 6C). The synergistic action of KCl and d,l-AP4 was also evident on evoked intracellular Ca2+ release. Thusd,l-AP4 or 30 mm KCl alone did not induce any significant Ca2+ response, but together evoked marked and reversible intracellular Ca2+ release (Fig.6D). These results indicated that coincident subliminal membrane depolarization and mGlu7 receptor activation were required for the synthesis of IP3 and intracellular Ca2+release. It is worth noting that a high concentration (100 mm) of KCl (equivalent to a steady-state depolarization to −10 mV) induced a similar IP response as a co-application of 100 mm KCl andd,l-AP4 (Fig. 6C). This result was consistent with the nonadditive inhibitory effects of a sustained depolarization to 0 mV and d,l-AP4 application on IBa (Figs. 5 and 6, B, open and filled circles). The present study shows that a long duration train of high frequency depolarization pulses, or a prolonged single steady state depolarization, can inhibit P/Q-type Ca2+ channels through a VGSC-Go protein-PLC-dependent pathway. These results provides the first electrophysiological evidence for a voltage-dependent activation of a G-protein via VGSCs, independently of Na+ flux. We also show that subliminal depolarizations were permissive for activation of this signaling cascade by the mGlu7 receptor (Fig.7). Inhibition of the P/Q-type Ca2+ channel induced by sustained membrane depolarization was different from the classical voltage-mediated inactivation of these channels, since P/Q-type Ca2+ channel inactivation has been previously described to be reversible, virtually abolished when Ba2+ was used as charge carrier, and should not depend on G protein or PKC activation (23Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Google Scholar, 24Lee A. Scheuer T. Catterall W.A. J. Neurosci. 2000; 20: 6830-6838Google Scholar). Our results were consistent with previous biochemical studies showing a voltage-dependent interaction of VGSC with Goprotein in rat brain synaptoneurosomes (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar). Indeed, R-DPI 201–106, a drug that blocks depolarization-induced activation of Goprotein in this preparation (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar), also blocked the depolarization-induced inhibition of Ca2+ channels in cerebellar granule cells. Although it is difficult to determine from the present study whether or not Go protein activation occurred during activation or inactivation of VGSCs, studies in synaptoneurosomes suggested that this may happen as long as depolarization lasts (15Anis Y. Nurnberg B. Visochek L. Reiss N. Naor Z. Cohen-Armon M. J. Biol. Chem. 1999; 274: 7431-7440Google Scholar). We show here that voltage-dependent activation of Go protein through VGSCs resulted in IP3 formation and intracellular Ca2+ release. Evidence for depolarization-induced IP3 formation has been previously reported in skeletal muscle (25Vergara J. Tsien R.Y. Delay M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6352-6356Google Scholar), and hyperpolarization has been suggested to reduce agonist-induced generation of IP3 in rabbit mesenteric artery (26Itoh T. Seki N. Suzuki S. Ito S. Kajikuri J. Kuriyama H. J. Physiol. 1992; 451: 307-328Google Scholar). Thus, the voltage-dependent activation of Go protein and IP3 synthesis observed here may not be specific for cerebellar granule cells. We found a synergistic action of VGSCs and mGlu7 receptors on IP3 formation and intracellular Ca2+ release. Voltage-dependent IP3-mediated Ca2+release has also been reported in coronary artery during stimulation of metabotropic cholinergic receptors (27Ganitkevich V. Isenberg G. J. Physiol. (Lond.). 1993; 470: 35-44Google Scholar). Moreover, during stimulation of metabotropic purinergic receptors, membrane depolarization can stimulate the release of Ca2+ from IP3-sensitive stores, in rat megakaryocytes. Consistent with our findings, the voltage sensor of this effect has been proposed to be upstream the purinergic receptors (28Mahaut-Smith M.P. Hussain J.F. Mason M.J. J. Physiol. (Lond.). 1999; 515: 385-390Google Scholar). Two alternative hypotheses can be proposed to explain the synergistic action of voltage and mGlu7 receptor in neurons. The mGlu7 receptors and VGSCs may share the same Go protein, or independently activate distinct Go proteins. Whatever the type of coupling to Go protein, a synergistic action of the receptor and VGSC was required to inhibit P/Q-type Ca2+channels. This provided a mechanism by which neuronal activity could promote glutamate-mediated metabotropic effects. It is likely that this mechanism occur at the axon terminal, since mGluR7 (6Kinzie J.M. Shinohara M.M. van den Pol A.N. Westbrook G.L. Segerson T.P. J. Comp. Neurol. 1997; 385: 372-384Google Scholar, 7Shigemoto R. Kulik A. Roberts J.D. Ohishi H. Nusser Z. Kaneko T. Somogyi P. Nature. 1996; 381: 523-525Google Scholar, 8Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J. Neurosci. 1997; 17: 7503-7522Google Scholar), PKC activity (29Rodriguez-Moreno A. Sistiaga A. Lerma J. Sanchez-Prieto J. Neuron. 1998; 21: 1477-1486Google Scholar), IP3-sensitive Ca2+ stores, and P/Q-type Ca2+ channels (13Dunlap K. Luebke J.I. Turner T.J. Trends Neurosci. 1995; 18: 89-98Google Scholar, 30Regehr W.G. Mintz I.M. Neuron. 1994; 12: 605-613Google Scholar, 31Takahashi T. Momiyama A. Nature. 1993; 366: 156-158Google Scholar, 32Turner T.J. Adams M.E. Dunlap K. Science. 1992; 258: 310-313Google Scholar) are present at presynaptic sites and control neurotransmitter release (10Lafon-Cazal M. Fagni L. Guiraud M.J. Mary S. Lerner-Natoli M. Pin J.P. Shigemoto R. Bockaert J. Eur. J. Neurosci. 1999; 11: 663-672Google Scholar, 11Lafon-Cazal M. Viennois G. Kuhn R. Malitschek B. Pin J.P. Shigemoto R. Bockaert J. Neuropharmacology. 1999; 38: 1631-1640Google Scholar). Thus high but not low frequency axonal discharges promotes coincident activation of presynaptic mGlu7 receptors by the neurotransmitter glutamate and action potential-induced activation of VGSCs, in the axon terminal. This coincident events would allow their synergistic inhibitory action on presynaptic P/Q-type Ca2+ channels and synaptic transmission. This hypothesis is consistent with the inhibitory effect of group III mGlu receptors on high but not low frequency synaptic transmission, observed in the rat locus coeuleus (14Dube G.R. Marshall K.C. J. Neurophysiol. 2000; 83: 1141-1149Google Scholar). It would also provide a mechanism by which mGlu7 receptor knockout mice develop epileptic seizures (33Masugi M. Yokoi M. Shigemoto R. Muguruma K. Watanabe Y. Sansig G. van der Putten H. Nakanishi S. J. Neurosci. 1999; 19: 955-963Google Scholar). We thank Dr. J. P. Pin and F. Ango for helpful discussions during preparation of the manuscript and Anne Cohen-Solal for technical assistance. We also thank Dr. J. Saugstad (Atlanta, GA) for the generous gift of the mGlu7a receptor cDNA and Dr. G. Romey (CNRS UPR411, Nice, France) for generous gift of theR- and S-enantiomers of DPI 201–106." @default.
• W2019934881 created "2016-06-24" @default.
• W2019934881 creator A5019805247 @default.
• W2019934881 creator A5033599123 @default.
• W2019934881 creator A5037127640 @default.
• W2019934881 creator A5045863654 @default.
• W2019934881 creator A5070954835 @default.
• W2019934881 date "2002-01-01" @default.
• W2019934881 modified "2023-10-12" @default.
• W2019934881 title "Permissive Effect of Voltage on mGlu 7 Receptor Subtype Signaling in Neurons" @default.
• W2019934881 cites W1600525795 @default.
• W2019934881 cites W1614343476 @default.
• W2019934881 cites W1639584526 @default.
• W2019934881 cites W1932328925 @default.
• W2019934881 cites W1946755838 @default.
• W2019934881 cites W1965649191 @default.
• W2019934881 cites W1968618064 @default.
• W2019934881 cites W1976564830 @default.
• W2019934881 cites W1980325410 @default.
• W2019934881 cites W1993779551 @default.
• W2019934881 cites W1999101834 @default.
• W2019934881 cites W2015242423 @default.
• W2019934881 cites W2035343100 @default.
• W2019934881 cites W2044151174 @default.
• W2019934881 cites W2050867525 @default.
• W2019934881 cites W2052255674 @default.
• W2019934881 cites W2063297445 @default.
• W2019934881 cites W2067935544 @default.
• W2019934881 cites W2070503276 @default.
• W2019934881 cites W2076980678 @default.
• W2019934881 cites W2077326004 @default.
• W2019934881 cites W2085691948 @default.
• W2019934881 cites W2089382485 @default.
• W2019934881 cites W2090102208 @default.
• W2019934881 cites W2091194839 @default.
• W2019934881 cites W2106042714 @default.
• W2019934881 cites W2117815723 @default.
• W2019934881 cites W2141479114 @default.
• W2019934881 cites W2141509024 @default.
• W2019934881 cites W2143601660 @default.
• W2019934881 cites W2148448646 @default.
• W2019934881 cites W2317324751 @default.
• W2019934881 doi "https://doi.org/10.1074/jbc.m109141200" @default.
• W2019934881 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11706005" @default.
• W2019934881 hasPublicationYear "2002" @default.
• W2019934881 type Work @default.
• W2019934881 sameAs 2019934881 @default.
• W2019934881 citedByCount "16" @default.
• W2019934881 countsByYear W20199348812013 @default.
• W2019934881 countsByYear W20199348812014 @default.
• W2019934881 countsByYear W20199348812017 @default.
• W2019934881 countsByYear W20199348812019 @default.
• W2019934881 countsByYear W20199348812021 @default.
• W2019934881 countsByYear W20199348812022 @default.
• W2019934881 countsByYear W20199348812023 @default.
• W2019934881 crossrefType "journal-article" @default.
• W2019934881 hasAuthorship W2019934881A5019805247 @default.
• W2019934881 hasAuthorship W2019934881A5033599123 @default.
• W2019934881 hasAuthorship W2019934881A5037127640 @default.
• W2019934881 hasAuthorship W2019934881A5045863654 @default.
• W2019934881 hasAuthorship W2019934881A5070954835 @default.
• W2019934881 hasBestOaLocation W20199348811 @default.
• W2019934881 hasConcept C15224491 @default.
• W2019934881 hasConcept C159047783 @default.
• W2019934881 hasConcept C169760540 @default.
• W2019934881 hasConcept C170493617 @default.
• W2019934881 hasConcept C185592680 @default.
• W2019934881 hasConcept C55493867 @default.
• W2019934881 hasConcept C62478195 @default.
• W2019934881 hasConcept C86803240 @default.
• W2019934881 hasConcept C95444343 @default.
• W2019934881 hasConceptScore W2019934881C15224491 @default.
• W2019934881 hasConceptScore W2019934881C159047783 @default.
• W2019934881 hasConceptScore W2019934881C169760540 @default.
• W2019934881 hasConceptScore W2019934881C170493617 @default.
• W2019934881 hasConceptScore W2019934881C185592680 @default.
• W2019934881 hasConceptScore W2019934881C55493867 @default.
• W2019934881 hasConceptScore W2019934881C62478195 @default.
• W2019934881 hasConceptScore W2019934881C86803240 @default.
• W2019934881 hasConceptScore W2019934881C95444343 @default.
• W2019934881 hasIssue "2" @default.
• W2019934881 hasLocation W20199348811 @default.
• W2019934881 hasOpenAccess W2019934881 @default.
• W2019934881 hasPrimaryLocation W20199348811 @default.
• W2019934881 hasRelatedWork W2067274453 @default.
• W2019934881 hasRelatedWork W2118512822 @default.
• W2019934881 hasRelatedWork W2152678253 @default.
• W2019934881 hasRelatedWork W2996143244 @default.
• W2019934881 hasRelatedWork W3193963519 @default.
• W2019934881 hasRelatedWork W4210463230 @default.
• W2019934881 hasRelatedWork W4231241026 @default.
• W2019934881 hasRelatedWork W4311090678 @default.
• W2019934881 hasRelatedWork W4382725342 @default.
• W2019934881 hasRelatedWork W3090890720 @default.
• W2019934881 hasVolume "277" @default.
• W2019934881 isParatext "false" @default.
• W2019934881 isRetracted "false" @default.
• W2019934881 magId "2019934881" @default.

• next