Ca2+ regulation of the carrier-mediated γ-aminobutyric acid release from isolated synaptic plasma membrane vesicles

Neuroscience Research 38 (2000) 385 – 395 www.elsevier.com/locate/neures

Ca2 + regulation of the carrier-mediated g-aminobutyric acid release from isolated synaptic plasma membrane vesicles J. Miguel Cordeiro a, Sandra M. Meireles b, M. Grac¸a P. Vale a, Catarina R. Oliveira a, Paula P. Gonc¸alves * b

a Centro de Neurocieˆncias, Uni6ersidade de Coimbra, 3004 -504 Coimbra, Portugal Centro de Biologia Celular, Departamento de Biologia, Uni6ersidade de A6eiro, 3810 -193 A6eiro, Portugal

Received 12 June 2000; accepted 22 August 2000

Abstract The regulation of the carrier-mediated g-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca2 + on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 mM GABA) and K+ depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca2 + (10 mM), the maximal velocity (Jmax) of K+ depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La3 + . In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca2 + was also observed by determining the kinetic parameters (K0.5 and Jmax) of influx into the SPM vesicles. Under these conditions, the Jmax of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca2 + ), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca2 + on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: GABA transporter; Ca2 + regulation; Synaptic plasma membrane

1. Introduction The g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system (Iversen, 1971). In physiological conditions, the fast synaptic clearance of GABA represents the most important function of the high affinity sodium-coupled transporter, located in the presynaptic Abbre6iations: GABA, g-aminobutyric acid; Jmax, maximal translocation velocity; Mes, 2[N-morpholino] ethanesulfonic acid; K0.5, GABA concentration to give half maximum uptake velocity; SPM, synaptic plasma membrane; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride. * Corresponding author. Tel.: + 351-23-4370766; fax: +351-234426408. E-mail address: [email protected] (P.P. Gonc¸alves).

plasma membrane (Iversen, 1971; Wood and Sidhu, 1986; Kanner, 1991; Amara and Kuhar, 1993). However, during the last 25 years, another function has been suggested on the basis of the involvement of the transporter in Ca2 + -independent cytoplasmic GABA release from nerve terminals (for review, see Bernarth, 1992; Richerson and Gaspary, 1997). It has been shown that, after nerve terminal depolarization associated with a sustained elevation in intracellular Na+ concentration, the GABA transporter operates in reverse, thereby mediating neurotransmitter release to the synaptic cleft (Pin and Bockaert, 1989; Turner and Goldin, 1989; During et al., 1995; Del Arco et al., 1998). In earlier experiments, we observed that GABA uptake by SPM vesicles is regulated by Ca2 + in a process which appears to involve the phosphorylation state of the trans-

0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 9 3 - 0

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porter (Gonc¸alves et al., 1997, 1999). Since reversion of the GABA transport system (Ca2 + -independent GABA release) has been evidenced in both normal and pathophysiological conditions (for review, see Meyer, 1991; Adam-Vizi, 1992; Attwell et al., 1993; Levi and Raiteri, 1993; Lester et al., 1996; Richerson and Gaspary, 1997), it appears interesting to investigate whether phosphorylation also regulates the GABA transporter activity when it mediates GABA release. The elucidation of the primary sequence of the GABA transporter through cDNA cloning has revealed the presence of consensus sequences for potential phosphorylation by protein kinases, including protein kinases A and C (Guastella et al., 1990; Nelson et al., 1990; Amara and Kuhar, 1993; Borden et al., 1992; Lo´pez-Corcuera et al., 1992; Liu et al., 1993; Bennett and Kanner, 1997; Clark 1997), suggesting that phosphorylation of amino acid residues may regulate the GABA transporter function. Furthermore, it has been reported that a variety of second messengers may trigger functional regulation of the GABA transporter through distinct pathways: by inducing interaction of the transporter with other synaptic proteins (Llina´s et al., 1985; Quick et al., 1997; Beckman et al., 1998), by modifying the number of functional surface transporters (Corey et al., 1994; Gomeza et al., 1994; Beckman et al., 1999; Bernstein and Quick, 1999) or by promoting post-translational modifications of the transporter molecule (Mager et al., 1993; Tian et al., 1994; Osawa et al., 1994; Sato et al., 1995). In fact, activators and inhibitors of protein kinase C and protein phosphatases alter the activity of a cloned rat brain GABA transporter expressed in Xenopus oocytes (Corey et al., 1994; Osawa et al., 1994). Sato et al. (1995) also demonstrated that this transporter, expressed in human embryonic kidney 293 cells, is downregulated by protein kinase C activation. Moreover, GABA uptake by striatal and cortical synaptosomes was decreased by treatment with protein phosphatase inhibitors or protein kinase A and C promoters (Osawa et al., 1994; Tian et al., 1994). Recently, we reported that the regulation of the GABA uptake by SPM vesicles depends on the activity of the internally localized calciumcalmodulin-dependent protein phosphatase (calcineurin), and that other phosphorylated sites, sensitive to protein phosphatases 1 and 2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively (Gonc¸alves et al., 1999). In this work, we studied the effect of Ca2 + on the carrier-mediated GABA release from SPM vesicles and we investigated the mechanism whereby protein phosphatase activities are involved in the Ca2 + modulatory action. We used isolated SPM vesicles, since this simple vesicle system is devoid of endogenous energy sources

and allows the study of GABA release in the absence of exocytotic neurotransmitter release (Gonc¸alves and Carvalho, 1995).

2. Materials and methods

2.1. Reagents The 4-amino-n-[2,3-3H]butyric acid ([3H]GABA), with a specific activity of 92.0 Ci per mmol, was purchased from Amersham International. Calyculin A, W-7 and ionomycin were supplied by Calbiochem-Novabiochem. Okadaic acid and cyclosporin A was obtained from Sigma–Aldrich. The filters used for SPM retention were obtained from Whatman Company.

2.2. Isolation of synaptic plasma membrane 6esicles The SPM vesicles were isolated from sheep brain cortex as earlier described (Gonc¸alves and Carvalho, 1997). After homogenization of the brain cortical grey matter, the homogenate was centrifuged at 900× g during 10 min and the resulting supernatant was centrifuged at 10 000× g during 20 min. After lysis of the pellet material (crude synaptosomal fraction) in hypotonic alkaline medium, followed by successive centrifugation at 8000× g (10 min) and 35 000× g (30 min), the resuspended pellet was centrifuged through a discontinuous Dextran T500 gradient for 2 h at 23 000 × g. The collected bands, containing the SPM, were diluted and centrifuged at 35 000× g for 30 min. Then, the pelleted vesicles were resuspended (5 mg protein per ml) and loaded with K+ by incubating for 30 min, at 30°C, in a medium containing 0.1 mM MgSO4 and 150 mM MES-potassium salt at pH 6.5. After the incubation period, SPM vesicles were centrifuged for 30 min at 35 000× g and the pellet was resuspended in the same buffer to give a final concentration of about 20 mg protein per ml. Finally, the preparation was divided in several aliquots, which were frozen in liquid nitrogen and stored at − 70°C. When required, the SPM vesicles were thawed at room temperature (RT). The protein was measured by the method of Gornall et al. (1949), using bovine serum albumin (BSA) as a standard.

2.3. Measurement of [ 3H]GABA uptake by SPM 6esicles The uptake of GABA by SPM vesicles was measured isotopically as earlier described (Gonc¸alves and Carvalho, 1997). The [3H]GABA uptake reactions were carried out at 30°C in a medium containing 150 mM NaCl, 10 mM HEPES-Na (pH 7.4), 50 mM EGTA and 2 mM ionomycin in the presence of increasing GABA concentrations (0.2–5.0 mM) supplemented with

J.M. Cordeiro et al. / Neuroscience Research 38 (2000) 385–395

[3H]GABA (0.25 mCi per ml). The reactions were initiated by adding SPM vesicles (final concentration 0.5 mg protein per ml) and they were stopped by rapid filtration of 500 ml aliquots through glass-fiber filters (Watman GF/B), prewashed with 5 ml of 150 mM NaCl. Then, the filters were washed with 10 ml of the same medium and they were plunged in scintillation cocktail (Universol™ ES) for further radioactivity measurement by liquid scintillation spectrometry. The values for [3H]GABA uptake by SPM vesicles were expressed as pmol/mg protein per s after subtraction of blank values obtained by filtering aliquots of reaction medium without SPM vesicles.

2.4. Measurement of [ 3H]GABA release by SPM 6esicles The release of [3H]GABA from SPM vesicles was studied by the superfusion technique described earlier (Gonc¸alves and Carvalho, 1997). The SPM vesicles were loaded with [3H]GABA during 2 min in the presence of 0.5 mM GABA as described above, except that ionomycin was absent. Using a peristaltic pump, the loaded SPM vesicles were drowed and spread on glass microfiber filters (Whatman GF/B) mounted in Millipore metal 13 mm Swinnex filter holders. The filters were continuously washed with a medium containing 150 mM NaCl, 10 mM HEPES-Na (pH 7.4), 50 mM EGTA, at a flow rate of 0.31 ml/min. After 8 min of superfusion, the medium was quickly substituted by the superfusion media indicated in the legends of the figures. Fractions were collected every minute directly into scintillation vials. The radioactivity was measured as described above for the uptake assays. The values for [3H]GABA release by SPM vesicles were expressed as dpm per fraction or as arbitrary units that were calculated using the following equation:

& x

GABA released (arbitrary units) =

a

3

n

Hx − 3Hb dt 3 Hb

where 3Hx corresponds to the dpm counted in fraction x and 3Hb corresponds to the extrapolated basal dpm level in the fraction x(a).

2.5. Measurement of

45

Ca 2 + uptake by SPM 6esicles

The uptake of Ca2 + by SPM vesicles was measured isotopically as described earlier (Coutinho et al., 1983). The 45Ca2 + uptake reactions were carried out at 30°C in a medium containing 150 mM KCl, 10 mM HEPESNa (pH 7.4), 50 mM EGTA and 59.6 mM 45CaCl2 (0.25 mCi/ml), in the presence of increasing LaCl3 concentrations (0–100 mM). The reactions were initiated by adding SPM vesicles (final concentration 0.5 mg protein per ml) and after 3 min, they were stopped by rapid filtration of 500 ml aliquots through glass-fiber filters

387

(Watman GF/B), prewashed with 5 ml of 320 mM sucrose, 10 mM Tris–HCl (pH 7.4) and 1 mM LaCl3. Then, the filters were washed with 10 ml of the same medium and they were plunged in scintillation cocktail (Universol™ ES) for further radioactivity measurement by liquid scintillation spectrometry. The values for 45 Ca2 + uptake by SPM vesicles were expressed as pmol per mg protein after subtraction of blank values obtained by filtering aliquots of reaction medium without SPM vesicles.

2.6. Treatment of the data GABA release values were calculated by using the Origin 6™ computer program and the Jmax corresponds to the slope of the resulting curves. All the results were treated statistically with the Graph-Pad-Prism™ computer program. Results are presented as mean9S.E.M. of the number of experiments indicated in the figures. Statistical significance was determined by means of an unpaired two-tailed Student’s t-test and P values are presented in the legends of the figures.

3. Results

3.1. Effect of Ca 2 + on the GABA release from synaptic plasma membrane 6esicles In earlier reports, we have demonstrated that GABA accumulation by SPM vesicles is down-regulated by intravesicular Ca2 + activation of the Ca-calmodulindependent protein phosphatase 2B (calcineurin) (Gonc¸alves et al., 1997, 1999). Since the electrogenic high affinity Na+-coupled GABA transport system also can translocate this neurotransmitter in the backward direction (Kanner and Kifer, 1981; Gonc¸alves and Carvalho, 1995), we studied the effect of Ca2 + on the GABA release from SPM vesicles. SPM vesicles were actively loaded with [3H]GABA during 2 min. Then, they were superfused 8 min with 150 mM NaCl, 50 mM EGTA and 10 mM HEPES-Na (pH 7.4) to remove the non accumulated [3H]GABA. As we can see in Fig. 1A, the replacement of the superfusion medium by a similar one, but containing 500 mM GABA or K+ in substitution of Na+, induces a massive efflux of this neurotransmitter from the vesicles. In these experiments, the effect of Ca2 + on the GABA release was studied by simultaneous introduction of 2 mM ionomycin and 59.67 mM CaCl2 ([Ca2 + ]free = 10 mM) into the different superfusion media, since maximal inhibitory effect of Ca2 + on the GABA uptake is achieved when free Ca2 + concentration inside the vesicles reaches  10 mM (Gonc¸alves et al., 1999). Under these conditions, we observed that Ca2 + (Fig. 1B) and ionomycin (results not shown) were unable to

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modify the GABA basal release (superfusion with 150 mM NaCl, 50 mM EGTA and 10 mM HEPES-Na at pH 7.4). Conversely, the K+ depolarization-induced GABA release (superfusion with 150 mM KCl, 50 mM EGTA and 10 mM HEPES-Na at pH 7.4) was significantly reduced by Ca2 + (Fig. 1B), whereas the GABA release by homoexchange (superfusion with 500 mM GABA, 150 mM NaCl, 50 mM EGTA and 10 mM HEPES-Na at pH 7.4) was only slightly affected (Fig. 1B). These results show that Ca2 + acts on the release of GABA from SPM vesicles in a way that depends on the mechanism of efflux that is activated under the various conditions tested (basal, homoexchange or K+ depolarization-induced release). Fig. 2 shows the time course of homoexchange-mediated GABA release by SPM vesicles under various experimental conditions. In all cases, the process exhibits the same pattern, with maximal release velocity (Jmax) between 3 and 5 min of superfusion and 7 min being the time required for efflux of the total amount of releasable GABA. The presence of Ca2 + outside the SPM vesicles (Fig. 2A) produced a statistical non-significant increment (30%) of the total amount of neurotransmitter released and, simultaneously, induced a proportional increase of the Jmax (from 1.8 to 2.1 a.u. per min). The observed stimulatory effect of extravesicular Ca2 + may reflect increased transport efficiency due to Ca2 + neutralization of membrane-surface negative charges of sialic acid molecules (Tapia and Salazar, 1989), rather than a direct effect of Ca2 + on the SPM GABA transport system. Actually, extravesicular Ca2 + up to 1 mM has no significant effect on the GABA

uptake by SPM vesicles and the EGTA removal of bound Ca2 + from the external membrane side results on a  12% reduction of SPM vesicle GABA retention capacity (Gonc¸alves et al., 1997). Furthermore, the superfusion of [3H]GABA loaded SPM vesicles with 500 mM GABA in the presence of 2 mM ionomycin, a selective Ca2 + ionophore that allows Ca2 + entry to the vesicles, decreased ( 20%) the earlier stimulatory effect of extravesicular Ca2 + (Fig. 2A and B), which seems to reflect a restrained inhibitory action of intravesicular Ca2 + on the GABA efflux from SPM vesicles by the homoexchange release pathway. Fig. 3 shows the Ca2 + effect on the K+ depolarization-induced release of GABA from SPM vesicles. In K+ depolarization conditions, Ca2 + content of SPM vesicles achieves 2.09 0.4 nmol/mg protein and the K+ depolarization-induced uptake of Ca2 + was completely prevented by 50 mM La3 + , the largest spectrum blocker of calcium channels (Fig. 3, insert). When Ca2 + enters into the vesicles in a depolarization-dependent manner, we observed a strong reduction (63%) of the total amount of GABA released (Fig, 3B) and this effect was abolished by superfusion in the presence of 50 mM La3 + (Fig. 3A). Moreover, 10 mM Ca2 + did not modify the amount of released GABA when NaCl was substituted with 300 mM sucrose in the superfusion medium (Fig. 3A). Additionally, Fig. 3B shows that the inhibitory effect of Ca2 + on the K+ depolarization-induced GABA release (63%) was even more pronounced (69%) in the presence of ionomycin, which enhanced by 1.9 fold the Ca2 + content of SPM vesicles (Fig. 3, insert). The fair correlation between the reduction de-

Fig. 1. Carrier-mediated [3H]GABA release from SPM vesicles in the absence (A) and in the presence of intravesicular Ca2 + (B). The SPM vesicles (0.5 mg protein per ml) actively loaded with [3H]GABA (incubation at 30°C, during 2 min, with [3H]GABA 0.5 mM, 150 mM NaCl, 50 mM EGTA and HEPES-Na, at pH 7.4) were superfused during 8 min with 150 mM NaCl, 50 mM EGTA and HEPES-Na (pH 7.4), as described in Section 2. Then, [3H]GABA release from SPM vesicles was induced by replacement of the superfusion medium with different superfusion media buffered with 10 mM Hepes-Na at pH 7.4 and containing 50 mM EGTA, in the absence (panel A) or in the presence of 2 mM ionomycin plus 10 mM free Ca2 + (panel B). – , plus 150 mM NaCl (basal release); – , plus 150 mM KCl (K+ depolarization-induced release); – , plus 500 mM GABA and 150 mM NaCl (homoexchange). The results are expressed in dpm/fraction and correspond to a representative experiment.

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Fig. 2. Effect of extravesicular (A) and intravesicular (B) Ca2 + on the homoexchange mediated release of [3H]GABA from SPM vesicles. Experimental conditions are similar to those described in the legend of Fig. 1. The [3H]GABA release from SPM vesicles loaded with [3H]GABA was induced by superfusion with 150 mM NaCl, 500 mM GABA, 50 mM EGTA and 10 mM HEPES-Na (pH 7.4) in absence (panel A) or in the presence of 2 mM ionomycin (panel B).  –, control conditions (without calcium); – , plus 10 mM free Ca2 + . The maximal velocities of [3H]GABA release are represented by Jmax. Values represent the integrated area of the released [3H]GABA as arbitrary U per min. The averages of six independent experiments are presented. The bars indicate S.E.M.

gree of the total GABA release and the reduction of the maximal release velocity (Jmax) appears to suggest that the modulatory role of Ca2 + is exerted at the level of the translocation reaction rate-limiting step. This hypothesis was properly checked by kinetic analysis of the GABA influx into the SPM vesicles. By Lineweaver-Burk analysis, depicted in Fig. 4, we calculated the values of maximal uptake velocity (Jmax = 25.59 1.9 pmol/s per mg protein) and those of GABA concentration to give half maximum uptake velocity (K0.5 = 3.19 0.3 mM) in the presence of intravesicular Ca2 + ([Ca2 + ]free =10 mM). By comparing these parameters with those (Jmax =17.4 92.1 pmol/s per mg protein and K0.5 =3.0 90.3 mM) obtained in control experiments (absence of Ca2 + ), we concluded that intravesicular Ca2 + altered the maximal uptake velocity without modify the affinity of the transport system for GABA. It appears, therefore, that intravesicular Ca2 + , at micromolar concentrations, reduces both the net uptake and the net release of GABA, by decreasing their maximal velocities of translocation across SPM.

3.2. Mechanism of the Ca 2 + inhibitory action on the K+ depolarization-induced GABA release from synaptic plasma membrane 6esicles It was earlier reported that the intravesicular Ca2 + inhibition of GABA accumulation by SPM vesicles is relieved by calcineurin inactivation and this event appears to be potentiated by inhibition of the protein phosphatase 1 or/and 2A activities (Gonc¸alves et al., 1999). In order to investigate the involvement of those protein phosphatase activities in Ca2 + inhibition of the

K+ depolarization-induced GABA release, we tested the effect of protein phosphatase inhibitors under conditions that allow the reversion of the GABA transport system. Fig. 5 shows that the micromolar Ca2 + inhibitory effect on GABA release was completely relieved by 100 mM cyclosporin A (a calcineurin inhibitor) (Fig. 5A) and by 10 mM W7 (a calmodulin antagonist) (Fig. 5B). When these compounds were introduced in the superfusion media, both the maximal velocity release and the total amount of [3H]GABA released from the SPM vesicles, in response to K+ depolarization, reached maximal values even in the presence of 10 mM Ca2 + (Figs. 3 and 5). Therefore, these results indicate that the underlying mechanism of Ca2 + inhibitory action on the SPM vesicle GABA release involves calcium/calmodulin protein phosphatase 2B (calcineurin) activation, as we previously observed under conditions that permit GABA uptake into SPM vesicles (Gonc¸alves et al., 1999). Interestingly, cyclosporin A and W7, in the absence of Ca2 + , did not significantly alter K+ depolarizationinduced release (Figs. 3 and 5), basal release or homoexchange mediated [3H]GABA efflux (results not shown) from preloaded SPM vesicles. This insensitivity to cyclosporin A and W7 of the GABA release from SPM vesicles, in the absence of Ca2 + , is in contrast to the significant changes produced by protein phosphatase inhibitors on GABA uptake carried out in the absence of added Ca2 + (Gonc¸alves et al., 1999), It appears, therefore, that the maximal GABA transporter activity, when it mediates neurotransmitter release, is not modified by the SPM dephosphorylation state.

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These observations, which reflect distinct modulation of the GABA transporter activity according to the main direction of GABA translocation across the SPM, were further ascertained by testing the effect of 100 nM okadaic acid and 2 nM calyculin A (protein phosphatase 1 and 2A inhibitors) on the GABA release by SPM vesicles. In all assayed conditions (basal, homoexchange-mediated and K+ depolarization-induced release), these protein phosphatase 1 and 2A inhibitors did not significantly modify neither the maximal release velocity nor the total amount of released neurotransmitter. In the case of GABA release from preloaded SPM vesicles in response to K+ depolarization (Fig. 6A), the presence of 100 nM okadaic acid and 2 nM calyculin A allowed to achieve release parameters (Jmax and total GABA released) values close to those obtained in control experiments (absence of protein phosphatase inhibitors) (Fig. 3A). Moreover, the strong reduction by Ca2 + of both the total amount of GABA released (50%) and the Jmax (67%) was also observed in the presence of okadaic acid and calyculin A (63 and 64% reduction for total released amount and Jmax, respectively), as earlier described in Fig. 3. Similarly, cyclosporin A also completely relieved the micromolar Ca2 + inhibition of the K+ depolarization-induced GABA release from SPM vesicles (Fig. 6B). In contrast to the ascertained regulatory mechanism of GABA uptake (Gonc¸alves et al., 1999), it appears that the regulation of the GABA release by calcium/calmodulin

activated protein phosphatase 2B is not potentiated by phosphorylation of protein phosphatase 1 and 2A sensitive sites, since only a non-statically significant reduction of the total amount of released GABA and of the maximal release velocity were observed when all the protein phosphatase inhibitors were simultaneously present in the superfusion media (Fig. 6). The results reported here indicate that the regulation of the SPM GABA transporter, when operating in the reverse mode (net GABA release), only requires phosphorylation of internally localized calcineurin-sensitive sites whereas, in normal mode (net GABA uptake), regulation also involves phosphorylation of the protein phosphatase 1 and 2A sensitive sites.

4. Discussion The results reported here clearly show that GABA release by SPM vesicles is differently regulated, according to the release mechanism that mediates GABA efflux (basal, homoexchange and K+ depolarization-induced release). Micromolar Ca2 + does not modify significantly the basal and homoexchange-mediated GABA release by SPM vesicles (Figs. 1 and 2), whereas the K+ depolarization-induced release of GABA was drastically diminished (69%) in the presence of 10 mM free Ca2 + inside the vesicles (Figs. 1 and 3), which was earlier demonstrated to be sufficient to induce maximal

Fig. 3. Effect of extravesicular (A) and intravesicular (B) Ca2 + on the K+ depolarization-induced [3H]GABA release from SPM vesicles. Experimental conditions are similar to those described in the legend of Fig. 1. The [3H]GABA release from SPM vesicles loaded with [3H]GABA was induced by superfusion with 150 mM KCl, 50 mM EGTA and 10 mM HEPES-Na (pH 7.4) (close symbols) or by superfusion with 320 mM sucrose, 50 mM EGTA and 10 mM HEPES-NA (pH 7.4) (open symbols). -, depolarization medium (without calcium); + – + , plus 10 mM free Ca2 + and 50 mM La3 + ; – , plus 10 mM free Ca2 + and 2 mM ionomycin; – , plus 10 mM free Ca2 + ; "– ", plus 2 mM ionomycin;  –, sucrose medium (without calcium);  –, plus 10 mM free Ca2 + . The maximal velocities of [3H]GABA release are represented by Jmax. Values represent the integrated area of the released [3H]GABA as arbitrary U per min. A dose-response curve for La3 + (0 – 100 mM) of the Ca2 + taken up by SPM vesicles in K+-depolarization conditions is present as an insert. The full line and the broken line represent the amount of Ca2 + retained by the SPM vesicles in the presence of 2 mM ionomycin and in 150 mM NaCl, 150 mM EGTA and 10 mM HEPES-Na (pH 7.4), respectively. The averages of six independent experiments are presented. The bars indicate S.E.M.

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Fig. 4. Kinetic analysis of the inhibitory Ca2 + effect on the [3H]GABA uptake by SPM vesicles. The uptake reactions were carried out at 30°C for 15 s, as described in Section 2. SPM vesicles (0.5 mg protein per ml) were incubated in a medium containing 150 mM NaCl, 50 mM EGTA, 2 mM ionomycin, 10 mM HEPES-Na (pH 7.4) and increasing concentrations (0.2–5.0 mM) of [3H]GABA. – , control conditions (absence of Ca2 + ); – , plus 10 mM free Ca2 + . Kinetic parameters (K0.5 and Jmax) were calculated by Lineweaver – Burk plotting of the values presented as insert. Vertical bars denote S.E.M. of the mean values of six independent experiments.

protein phosphatase 2B activation and GABA uptake inhibition (Kincaid and Vaughan, 1986; Armstrong, 1989; Gonc¸alves et al., 1999).

391

The involvement of protein phosphatase 2B in mediating the inhibitory effect of Ca2 + on the GABA release by SPM vesicles in response to K+ depolarization was supported by the observations that it is completely relieved in the presence of the calmodulin antagonist, W-7 (Hidaka et al., 1981) or of the protein phosphatase 2B inhibitor, cyclosporin A (Swanson et al., 1992; Guerini, 1997) (Fig. 5), even when inhibitors of protein phosphatase 1 and 2A are present in the reaction medium (Fig. 6). Moreover, the insensitivity of GABA release to 100 nM okadaic acid and 2 nM calyculin A seems to rule out the involvement of protein phosphapases 1 and 2A in potentiating the protein phosphatase 2B modulatory action on the carrier-mediated GABA release, as we observed when GABA transporter mediates GABA uptake (Gonc¸alves et al., 1999). Sim et al. (1993) proposed protein phosphatase 1 and 2A-mediated up-regulation of GABA release from rat forebrain synaptosomes, since treatment with 1 mM okadaic acid elevated basal release of endogenous GABA in the presence of 1.2 mM Ca2 + . Okadaic acid, at micromolar concentrations, is a large spectrum protein phosphatase inhibitor (Bialojan and Takai, 1988; Wera and Hemmings, 1995) that seems to promote, at least in the presence of Ca2 + , a marked elevation in the phosphorylation of many synaptosomal proteins, relatively to that observed when nerve terminals respond to K+ depolarization (Sim et al., 1993). It was earlier established that protein kinase A and C promoters can enhance GABA uptake by isolated synaptosomes (Osawa et al., 1994; Tian et al., 1994), in agreement with similar effects in reconstitution systems expressing the cloned rat GABA transporter (GAT1)

Fig. 5. Effect of cyclosporin A (A) and W-7 (B) on the Ca2 + -mediated inhibition of the K+ depolarization-induced [3H]GABA release from SPM vesicles. Experimental conditions are similar to those described in the legend of Fig. 3. The [3H]GABA release from SPM vesicles loaded with [3H]GABA was induced by superfusion with 150 mM KCl, 50 mM EGTA and 10 mM HEPES-Na (pH 7.4).  – , plus 100 mM cyclosporin A (panel A) or 10 mM W-7 (panel B); – , plus 10 mM free Ca2 + and 100 mM cyclosporin A (panel A) or 10 mM W-7 (painel B); -, plus 10 mM free Ca2 + . The maximal velocitys of [3H]GABA release are represented by Jmax. Values represent the integrated area of the released [3H]GABA as arbitrary U per min. The averages of six independent experiments are presented. The bars indicate S.E.M.

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Fig. 6. Ca2 + -mediated inhibition of the K+ depolarization-induced [3H]GABA release from SPM vesicles in the presence of protein phosphatases 1 and 2A inhibitors: relieve by cyclosporin A. Experimental conditions are similar to those described in the legend of Fig. 3. The [3H]GABA release from SPM vesicles loaded with [3H]GABA was induced by superfusion with 150 mM KCl, 50 mM EGTA and 10 mM HEPES-Na (pH 7.4) containing 100 nM okadaic acid and 2 nM calyculin A (protein phosphatases 1 and 2A inhibitors), in the absence (panel A) or in the presence of 100 mM cyclosporin A (panel B).  – control conditions (without Ca2 + ); – , plus 10 mM free Ca2 + . The maximal velocities of [3H]GABA release are represented by Jmax. Values represent the integrated area of the released [3H]GABA as arbitrary U per min. The averages of six independent experiments are presented. The bars indicate S.E.M.

(Corey et al., 1994; Osawa et al., 1994; Sato et al., 1995). In our experiments with SPM vesicles, it was possible to demonstrate that calcium/calmodulin activation of protein phosphatase 2B is responsible for down-regulation of carrier-mediated GABA release in response to K+ depolarization, whereas it does not modify homoexchange or basal release. It is interesting to note that the protein phosphatase modulatory action seems to reflect a reduction of the maximal release velocity (Fig. 3), suggesting that phosphorylation of calcineurinsensitive sites may be important for the translocation reaction rate-limiting step. This conclusion is also supported by the observation that maximal velocity for net GABA uptake by SPM vesicles is slowed by intravesicular micromolar Ca2 + (Fig. 4), whereas the homoexchange-mediated release, that proceeds with a maximal velocity 1.5-fold higher than that of net release (Figs. 3 and 4), is not significantly modified either by Ca2 + or by W7 and cyclosporin A. Taking in consideration that GAT-1 seems to be responsible for more than 95% of synaptosomal GABA uptake (Sutch et al., 1999), it appears that the different sensitivities of the two types of GABA release by SPM vesicles (homoexchange and K+ depolarization-induced release) reflect different sequence of GABA translocation reaction steps by GAT-1 rather than participation of several different transporters (GAT-2, GAT-3 or BGT-1) (Eriksson et al., 1999; Soudijn and van Wijngaarden, 2000). In fact, according to the predictions of the developed models for the GABA transport system at the nerve terminals (Wheeler, 1984; Kanner, 1989;

Lester et al., 1996), it is plausible to assume that the characteristics and the sequence of the translocation reaction steps are distinct when the transport system mediates either net uptake, homoexchange or net release. The GABA transport, energized by the Na+ concentration gradient across the plasma membrane, is electrogenic and voltage-dependent. These characteristics allowed the study of functional properties of GAT1 expressing systems by electrophysiological approaches, which ascertained some individual steps in the transport cycle. In the presence of saturating GABA concentrations, when the transporter is fully occupied by two Na+ ions and one Cl− ion, the overall transport process has already undergone its rate-limiting step before GABA binding and subsequent translocation (Kanner, 1989; Cammack et al., 1994; Mager et al., 1996). In our experimental conditions, this situation corresponds to the homoexhange-mediated GABA release (the steadystate distribution of the transporter in the GABA-receptive state is very high), which is insensitive to calcium/calmodulin activation of protein phosphatase 2B (Fig. 1) and occurs at a maximal release velocity (Fig. 2) higher than that observed for K+ depolarization-induced release (Fig. 3). The hypothesized GABA transport rate-limiting step appears to involve the conformational change of the transporter, which depends on the Na+ and/or Cl− binding at saturating GABA concentrations (Mager et al., 1996; Golovanevsky and Kanner, 1999), whereas at low GABA concentrations and high negative potentials, the voltage-independent interaction between GABA and the transporter is suffi-

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cient to limit the velocity of the transport (Mager et al., 1993). Probably, when SPM vesicles release GABA in response to K+ depolarization, the transporter molecule must also undergo conformational changes in order to facilitate the GABA efflux from the intra to the extravesicular space and this process is slower when SPM protein phosphatase 2B is activated by calcium/ calmodulin (Figs. 3 and 5). In conclusion, these results suggest that calcium/calmodulin activated protein phosphatase 2B activity is involved in the regulation of the SPM GABA transporter, and that maximal net translocation velocity requires phosphorylation of calcineurinsensitive sites internally localized. Thus, the operativity of the GABA transporter appears to be regulated by its state of phosphorylation-dephosphorylation, in agreement with our earlier results showing that the maximal

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active GABA transport by SPM vesicles, in the presence of low GABA concentrations and optimal electrochemical ionic gradients, requires two types of phosphorylated sites: internal phosphorylated sites, which are sensitive to calcineurin, and phosphorylated sites which are sensitive to protein phosphatases 1 and/or 2A (Gonc¸alves et al., 1999). The modulatory role of phosphorylation/dephosphorylation processes in the net GABA fluxes across the presynaptic membrane may contribute to shape the time course and spatial extent of synaptic transmission by modifying the turnover rate of individual transporter molecules. The calcineurin-mediated Ca2 + effect on GABA translocation rate across SPM is represented in Fig. 7. During GABA exocytosis, calcineurin activation by increased Ca2 + influx should inhibit the carrier-

Fig. 7. Schematic representation of the modulatory role of dephosphorylation processes on the SPM GABA transporter activity during exocytotic phase (A), reuptake phase (B), and resting phase (C). D, putative regulatory site sensitive to protein phosphatase 2B (PP2B); , down-regulation by dephosphorylation;  , maximal uptake; ¡, maximal release; M, maximal homoexchange; O, reduced uptake; n, reduced release. Explanation is given in the text.

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mediated GABA release by dephosphorylating the internal regulatory sites (Fig. 7A). On the other hand, maximal GABA transporter activation should be suitable for GABA recovering into the pre-synaptic nervous terminal after the exocytotic release, when the voltage-dependent Ca2 + influx was blocked (Fig. 7B). Finally, the resting phase, at which the GABA gradient must be maintained, should correspond to the steadystate of the process that is not sensitive to dephosphorylation of the internal calcineurin sensitive sites (Fig. 7C). The carrier-mediated release of GABA appears to contribute for generating increased inhibitory tone at the synapses during periods of intense neuronal and glial excitation, which occurs before seizure generation (During et al., 1995). However, as the intracellular Ca2 + concentrations increase during prolonged excitation (\ 1 mM) (Tse and Tse, 1999), the carrier-mediated release of GABA is reduced with consequent decrease of the inhibitory tone that characterize the development of epileptogenic states. These assumptions support the idea that Ca2 + regulation of the carriermediated GABA fluxes at the synapse level may be implicated in shaping the GABAergic signaling in normal and pathophysiological conditions. Acknowledgements This research was supported by PRAXIS XXI. References Adam-Vizi, V., 1992. External Ca2 + -independent release of neurotransmitters. J. Neurochem. 58, 395–405. Amara, S.G., Kuhar, M.J., 1993. Neurotransmitter transporters: recent progress. Annu. Rev. Neurosci. 16, 73–93. Armstrong, D.L., 1989. Calcium channel regulation by calcineurin, a Ca2 + -activated phosphatase in mammalian brain. TINS 12, 117 – 122. Attwell, D., Barbour, B., Szatkowski, M., 1993. Nonvesicular release of neurotransmitter. Neuron 11, 401–407. Beckman, M.L., Bernstein, E.M., Quick, M.W., 1998. Protein kinase C regulates the interaction between a GABA transporter and syntaxin 1A. J. Neurosci. 18, 6103–6112. Beckman, M.L., Bernstein, E.M., Quick, M.W., 1999. Multiple G protein-coupled receptors initiate protein kinase C redistribution of GABA transporters in hippocampal neurons. J. Neurosci. 19RC9, 1 – 6. Bennett, E.R., Kanner, B.I., 1997. The membrane topology of GAT1, a (Na+ + Cl−)-coupled g-aminobutyric acid transporter from rat brain. J. Biol. Chem. 272, 1203–1210. Bernarth, S., 1992. Calcium-independent release of amino acid neurotransmitter: fact or artifact? Prog. Neurobiol. 38, 57–91. Bernstein, E.M., Quick, M.W., 1999. Regulation of g-aminobutyric acid (GABA) transporters by extracellular GABA. J. Biol. Chem. 274, 889 – 895. Bialojan, C., Takai, A., 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J. 256, 283–290.

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