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Regulation of [γ-3H]aminobutyric acid transport by Ca2+ in isolated synaptic plasma membrane vesicles
Molecular Brain Research 51 Ž1997. 106–114
Research report
Regulation of w g- 3 Hxaminobutyric acid transport by Ca2q in isolated synaptic plasma membrane vesicles Paula P. Gonc¸alves b
a,)
, Arselio ´ P. Carvalho b, M. Grac¸a P. Vale
b
a Departamento de Biologia, UniÕersidade de AÕeiro, 3810 AÕeiro, Portugal Centro de Neurociencias, Departamento de Zoologia, Faculdade de Ciencias, UniÕersidade de Coimbra, Coimbra, Portugal ˆ ˆ
Accepted 25 June 1997
Abstract We studied the effect of Ca2q on the transport of the g-aminobutyric acid ŽGABA. by synaptic plasma membrane ŽSPM. vesicles isolated from sheep brain cortex and observed that intravesicular Ca2q inhibits the w 3 HxGABA accumulation in a concentration-dependent manner. This inhibitory effect of Ca2q exhibited two distinct components: one in the micromolar range of Ca2q concentration, and the other in the millimolar range. Previous EGTA washing of the membranes, or incorporation of trifluoperazine into the vesicular space reduced the inhibitory action of Ca2q, particularly at low Ca2q Ž1–5 mM.. Okadaic acid Ž1 mM. also relieved the Ca2q inhibition at low, but not at high Ca2q concentrations Ž1 mM., whereas the calpain inhibitor I did not alter the effect of the low Ca2q, but it partially reduced Ž; 28%. the effect of Ca2q in the millimolar range. The results indicate that the GABA transporter is regulated by low Ca2q concentration ŽmM. and probably its effect is mediated by the ŽCa2q Ø calmodulin.-stimulated phosphatase 2B Žcalcineurin.. In contrast, the GABA uptake inhibition observed at high Ca2q concentrations Ž1 mM. is less specific, and probably it is partially related to the proteolytic activity of membrane bound calpain II. q 1997 Elsevier Science B.V. Keywords: GABA transport; Synaptic plasma membrane; Ca2q regulation
1. Introduction In neuronal synapses, release of g-aminobutyric acid ŽGABA. has been described either by Ca2q-induced exocytosis or by Ca2q-independent reversal of the carrier-mediated transport system at the level of the plasma membrane w40x. We observed previously that the Ca2q-independent process of GABA release by isolated synaptic plasma membrane ŽSPM. vesicles in fact is inhibited by Ca2q under conditions of Kq-induced depolarization, which indicates that Ca2q may modulate the activity of the GABA transporter w23x. The inhibitory action of Ca2q on the carrier-mediated release of neurotransmitters is also supported by the fact that decrease of extracellular Ca2q enhances neurotransmitter release induced by Kq-depolari-
Abbreviations: w 3 HxGABA, w g- 3 Hxaminobutyric acid; Mes, 2-Ž Nmorpholino.-ethanesulfonic acid; SPM, synaptic plasma membraneŽs.. ) Corresponding author. Fax: q351 Ž34. 26-408. 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 2 3 - 4
zation w10,14,37,38x and electrically evoked release w7,13,48x. Furthermore, in platelets, intracellular Ca2q was reported to regulate the uptake of 5-hydroxytryptamine w36x and, in intact retinal cells, Ca2q was shown to inhibit the glutamate transporter by a process which involves production of arachidonic acid due to Ca2q activation of phospholipase A 2 w15x. On the other hand, the results of Tapia and co-workers w45x showed that neutralization of negative surface charges by extracellular Ca2q is necessary for GABA uptake by synaptosomes. It appears, therefore, that Ca2q compartmentation is important for its effects at the level of the neurotransmitter transporters. In spite of these findings, the mechanism involved in the Ca2q regulation of the neurotransmitter transporters remains to be clarified. In presynaptic membranes, phosphorylationrdephosphorylation has been reported to alter the efficiency of neurotransmitter release w33,41,43x. The possibility of GABA transport modulation by second messengers has been considered since 1990, when Guastella et al. w25x, by cloning the cDNA for the rat brain GABA transporter and by expressing the protein sequence in
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oocytes, observed that this protein has up to one putative protein kinase A and seven putative protein kinase C phosphorylation sites. Indeed, it was reported that activators and inhibitors of protein kinase C and protein phosphatases regulate the activity of a cloned rat brain GABA transporter expressed in Xenopus oocytes w12x. More recently, it was demonstrated that this transporter expressed in human embryonic kidney 293 cells is downregulated by protein kinase C activation w39x. In this work, we studied the effect of Ca2q on the GABA transport by SPM vesicles, and the mechanism whereby Ca2q modulates the transporter. We used isolated plasma membranes in our study since this simple vesicle system allows easy changes in the ionic gradients across its membranes and GABA transport machinery can be studied in the absence of exocytotic release of GABA w23x. Our results show that Ca2q regulates the GABA transporter when it works in the forward direction ŽGABA uptake. and this effect is modulated by calmodulin in a process which appears to involve calcineurin.
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2.3. Incorporation of Ca 2 q and calmodulin antagonists into synaptic plasma membrane Õesicles The effect of intravesicular Ca2q on the process of GABA accumulation by SPM vesicles was studied by carrying out reactions of w 3 HxGABA uptake in the presence of 2 mM ionomycin which permits Ca2q entry. On the other hand, the calmodulin antagonists, trifluoperazine and compound 48r80, were incorporated into the vesicles by the freezing and thawing method w35x. Thus, SPM vesicles Ž20 mg proteinrml. were frozen and thawed in the presence of 800 mM trifluoperazine or 780 mgrml compound 48r80 before the initiation of the GABA uptake reaction. After dilution of the membranes in the reaction mixture, the final concentrations of the drugs were about 40 mM and 29 mgrml, respectively. Ionomycin was suitable for Ca2q incorporation into the vesicles, since, in contrast to the freeze–thaw method, it permits Ca2q entry at the time of reaction, avoiding activation of Ca2q proteases due to a delayed contact with Ca2q before starting the GABA uptake reaction.
2. Materials and methods
2.4. Measurement of [ 3 H]GABA uptake by SPM Õesicles
2.1. Materials
The w 3 HxGABA uptake was assayed, at 308C, in a reaction mixture containing 150 mM NaCl, 10 mM HEPES-Na ŽpH 7.4., GABAq w 3 HxGABA Ž0.5 mM, 0.25 mCirml., and 5 mM or 1 mM Ca2q in the presence or absence of ionomycin. The reactions were initiated by addition of the Mes-potassium-loaded vesicles to a final protein concentration of 0.5 mgrml. At several time intervals, the reactions were stopped by rapid filtration of 0.5 ml through glass-fiber filters ŽWhatman GFrB. prewashed with 5 ml of 150 mM NaCl. After filtration of the samples, the filters were washed with 10 ml of the same NaCl solution and the amount of w 3 HxGABA taken up was determined by liquid scintillation spectrometry. The radioactivity on the filters was measured in 8 ml of scintillation cocktail Žcomposition per litter of toluene: 7.3 g of 2,5-diphenyloxazole ŽPPO.; 176 mg of p-bis 2X-Žphenyloxazolyl.benzene ŽPOPOP. and 250 ml of Triton X-100., utilizing a Packard Tri-carb 460 CD liquid scintillation counter. In some assays, the reaction medium contained 2.5 mM valinomycin, 20–100 mM trifluoperazine, 39–80 mgrml compound 48r80, 0.001–10 mM okadaic acid or 0.5–5 mM calpain inhibitor I. Some experiments were carried out with SPM vesicles previously incorporated with trifluoperazine or compound 48r80 as described above.
The 4-amino-n-w2,3- 3 Hxbutyric acid Žw 3 HxGABA., with a specific activity of 92.0 Cirmmol, was obtained from Amersham International. Valinomycin and compound 48r80 were purchased from Sigma-Aldrich, whereas trifluoperazine was supplied by SmithKline and French. The ionomycin and calpain inhibitor I were obtained from Calbiochem-Novabiochem, whereas okadaic acid was supplied by Research Biochemicals International. All reagents were analytical grade. The filters used for SPM retention were obtained from Whatman Co.. 2.2. Isolation of synaptic plasma membrane Õesicles The SPM vesicles were isolated from sheep brain cortex as previously described w21x. After isolation, the vesicles were loaded with Kq by incubating 30 min, at 308C, in 150 mM Mes-potassium salt ŽpH 6.5. and 0.1 mM MgSO4 , at a protein concentration of 5 mgrml. They were then centrifuged for 30 min at 35 000 = g and resuspended in the same buffer to a final protein concentration of about 20 mgrml. The analysis of protein was performed by the method of Gornall w24x and the material was divided into aliquots which were rapidly frozen in liquid nitrogen and stored at y708C. When required, the SPM vesicles were thawed at room temperature. In some experiments, SPM vesicles were washed with 0.1 mM EGTA to remove membrane bound calmodulin. After centrifugation, the trapped EGTA in the pellets was eliminated during the washing in the Kq-loading medium as described above.
2.5. Measurement of the membrane electric potential The Dc of the vesicles were determined at 308C by using a tetraphenyl phosphonium ŽTPPq. selective electrode in a medium containing 8 mM TPPq and 1 mg of
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protein. The Dc of the SPM vesicles was calculated from the accumulation of TPPq according to the following equation w28x:
Dc s 2.3 RTrF log Ž ÕrV . y 2.3 RTrF log Ž 10 F r D E r2.3RT y 1 . The external volume Ž V . is equal to 1 ml and the internal average volume Ž Õ . of SPM vesicles is 3.1 " 0.7 mlrmg protein as determined by the method of Chan et al. w11x. The measured Dc were corrected considering that the Dc of the vesicles is zero when 2.5 mM valinomycin and 150 mM KCl are present in the external medium.
3. Results 3.1. Effect of Ca 2 q on the GABA uptake by synaptic plasma membrane Õesicles The effects of Ca2q on the GABA uptake by SPM vesicles were investigated either when Ca2q was present at the external side of the membrane or when it was localized intravesicularly. Fig. 1A shows the time course of w 3 HxGABA uptake by SPM vesicles under different conditions of Ca2q concentration and Ca2q compartmentation. We observed that extravesicular Ca2q has no significant effect on the process of w 3 HxGABA uptake by SPM vesicles which maximally accumulated 125 pmol GABArmg protein at 2.5 min of reaction. A slight decrease Ž; 12%. was observed in the presence of 0.5 mM EGTA which removes the Ca2q bound to the external side of the membranes. In contrast, in the presence of ionomycin, when Ca2q entered the
vesicles, it inhibited the w 3 HxGABA uptake. The w 3 HxGABA accumulation was maximal Ž99.4 pmol w 3 HxGABA taken uprmg protein. in the presence of EGTA, which indicates that even contaminating Ca2q Žabsence of EGTA. was sufficient to reduce Ž; 22%. the SPM w 3 HxGABA uptake Ž77.5 pmolrmg protein.. In studies of w 3 HxGABA uptake by SPM as a function of Ca2q concentration ŽFig. 1B., we observed that, in the presence of 2 mM ionomycin, the process exhibits two distinct phases: in the micromolar range, Ca2q had a small but significant inhibitory effect on the w 3 HxGABA uptake Ž; 30%. ŽFig. 1B., whereas in the millimolar range Žabove 0.5 mM., w 3 HxGABA accumulation sharply declined, indicating that, under these conditions, intravesicular Ca2q had a strong inhibitory effect, which reached about 70% at 2 mM Ca2q. Control experiments performed in the absence of ionomycin showed no effect on w 3 HxGABA uptake ŽFig. 1B.. Since membrane depolarization induces w 3 HxGABA release w4–8,22,23,44x, we tested whether, under our experimental conditions, membrane potential variations which could be associated with the Ca2q entry into the vesicles would cause inhibition of w 3 HxGABA accumulation. Fig. 2 shows that in a valinomycin-containing medium, where the membrane potential was determined essentially by the potassium ion gradient, the Ca2q inhibitory effects persisted. Indeed, in the presence of ionomycin either contaminant Ca2q Ž; 1.5 mM. in the medium, or 1 mM Ca2q reduced the amount of w 3 HxGABA taken up by the vesicles. It is evident that in the presence of valinomycin, both components of the Ca2q inhibition remained, although the absolute values for w 3 HxGABA accumulated were higher than those observed in the absence of the Kq ionophore.
Fig. 1. Effect of Ca2q on the w 3 HxGABA uptake by SPM vesicles. The SPM vesicles Ž0.5 mg proteinrml. were incubated at 308C in a solution containing 150 mM NaCl, 10 mM HEPES-Na ŽpH 7.4. and 0.5 mM w 3 HxGABA Žsodium-medium. under various experimental conditions. A: time course of w 3 HxGABA uptake in the presence or in the absence of ionomycin. The reactions were carried out in the absence Ž`. and in the presence of 1 mM CaCl 2 Žv ., 0.5 mM EGTA Ž^., 0.5 mM EGTA plus 2 mM ionomycin Ž'., 2 mM ionomycin ŽI., 5 mM CaCl 2 plus 2 mM ionomycin ŽB. or 1 mM CaCl 2 plus 2 mM ionomycin Žl.. The reactions were stopped by filtering 0.5 ml aliquots and the filters radioactivity was measured as described in Section 2. B: w 3 HxGABA uptake as a function of Ca2q concentration. The reactions were carried out for 2.5 min in the absence Ž`. and in the presence of 2 mM ionomycin Žv .. Vertical bars denote S.D. of the mean value of 5–8 separate determinations.
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Fig. 2. Effect of valinomycin on the inhibition of w 3 HxGABA uptake by Ca2q. The reactions were carried out as described in Fig. 1, in the absence ŽA. or in the presence ŽB. of 2.5 mM valinomycin, so that the membrane potential was essentially determined by the Kq gradient. Sodium-medium Ž`.; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA Žv .; sodium-medium plus 2 mM ionomycin Ž^. and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Ž'.. Inset shows the Dc of the SPM vesicles in the absence and in the presence of 2.5 mM valinomycin. Vertical bars denote S.D. of the mean value of 3–5 separate determinations.
This suggests that the electric potential of the membrane is important to determine the extent of the w 3 HxGABA transport but the inhibitory effect of Ca2q persisted independently of the membrane potential magnitude Žy38.6 " 0.7 mV or y25.3 " 1.3 mV in the presence or absence of valinomycin, respectively ŽFig. 2, inset... However, the effect of high Ca2q concentrations ŽmM. was reduced by
about 17% in the presence of valinomycin, whereas that of low Ca2q concentrations ŽmM. was slightly altered Ž5%.. 3.2. Mechanisms inÕolÕed in the Ca 2 q effects on the GABA uptake by synaptic plasma membrane Õesicles We observed that the magnitude of the Ca2q inhibitory effects was reduced when calmodulin antagonists, trifluop-
Fig. 3. Ca2q inhibition of w 3 HxGABA uptake by SPM vesicles containing calmodulin antagonists. The assays were performed as described in Fig. 1. For incorporation of the drug into the vesicles, the SPM vesicles Ž10 mg proteinrml. were freeze–thawed in the presence of 800 mM trifluoperazine or 780 mgrml compound 48r80 before starting the w 3 HxGABA uptake reaction. The final concentrations of trifluoperazine and compound 48r80 in the reaction medium were reduced to 40 mM and 29 mgrml, respectively. The reactions were carried out for 2.5 min under the various experimental conditions. Histograms show from left to right: sodium-medium Žwhite bar.; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA; sodium-medium plus 2 mM ionomycin; sodium-medium plus 2 mM ionomycin and 5 mM CaCl 2 ; and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Žblack bar.. Vertical bars denote S.D. of the mean value of 3–6 separate determinations. The inset shows the percentage of w 3 HxGABA uptake inhibition by Ca2q.
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erazine or compound 48r80, were incorporated into the vesicles ŽFig. 3.. This was particularly evident for the inhibition observed in the micromolar range of Ca2q concentration. The results show that the inhibitory effect of micromolar Ca2q concentrations was 68% reduced by trifluoperazine and completely eliminated by compound 48r80, whereas the inhibitory effect of millimolar Ca2q concentrations was only reduced by 16% or 34% in the presence of intravesicular trifluoperazine or compound 48r80, respectively ŽFig. 3.. Under the experimental conditions used, the trifluoperazine, per se, did not reduce the GABA uptake process. Instead, experiments performed at increasing trifluoperazine concentrations showed that incorporation of the drug into the vesicles promoted a slight enhancement of the w 3 HxGABA accumulation Žresults not shown.. In control experiments, we observed that the simple addition of this drug to the reaction medium did not alter significantly the normal w 3 HxGABA uptake process. Furthermore, the inhibitory effect of intravesicular Ca2q ŽmM or mM. on the w 3 HxGABA uptake was not significantly altered when trifluoperazine was only present outside the vesicles Žresults not shown.. Since similar results of Ca2q effects reduction were obtained with the other calmodulin antagonist, compound 48r80 ŽFig. 3., it appears that a calmodulin-dependent mechanism, localized at the internal side of the membrane, is responsible for the Ca2q inactivation of the GABA transporter in sheep brain SPM vesicles. Compound 48r80 antagonizes the calmodulin function with great specificity w17x. However, its utilization in SPM studies requires appropriate controls since the drug, by increasing membrane permeability to Kq w29x, probably depolarizes the membrane during the
phase of incorporation and, per se, reduces the GABA uptake. Conversely, when it acts extravesicularly, it increases GABA uptake probably by inducing membrane hyperpolarization associated to rapid Kq efflux Žresults not shown.. The observation that the inhibitory effect of micromolar Ca2q was partially relieved by calmodulin antagonists incorporated inside the vesicles strongly suggests that the Ca2q effect may be calmodulin mediated. We further tested this idea by studying the Ca2q effects on the GABA uptake by SPM vesicles previously washed with 0.1 mM EGTA to deplete them of intrinsic calmodulin. We observed that, as in native membranes, Ca2q inhibited the GABA uptake by EGTA-washed vesicles when it entered the vesicles in the presence of ionomycin ŽFig. 4.. It is of interest to note that the inhibitory effect of micromolar Ca2q concentrations was reduced by about 48% in EGTA-washed membranes, whereas the inhibitory effect of millimolar Ca2q concentrations was reduced by only about 7%, as compared to the inhibitory effects of intravesicular Ca2q in native membranes ŽFig. 4, inset.. Furthermore, we also observed that membrane EGTA washing increased the total amount of w 3 HxGABA accumulated either in the absence Ž179 pmolrmg protein per 2.5 min., or in the presence Ž135 pmolrmg protein per 2.5 min. of ionomycin, as compared to the values Ž120 pmolrmg protein per 2.5 min and 78 pmolrmg protein per 2.5 min. obtained for the native membranes under the same experimental conditions, respectively ŽFig. 4.. The results of kinetic studies, depicted in Fig. 5, showed that EGTA washing of the membranes altered the values of Jmax and K 0.5 for the w 3 HxGABA uptake reaction. By
Fig. 4. Comparison between the Ca2q effects on the w 3 HxGABA uptake by native and EGTA-washed SPM vesicles. The reactions were carried out for 2.5 min under the various experimental conditions. Histograms show from left to right: sodium-medium Žwhite bar.; sodium-medium plus 1 mM CaCl 2 ; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA; sodium-medium plus 2 mM ionomycin; sodium-medium plus 2 mM ionomycin and 5 mM CaCl 2 ; and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Žblack bar.. Vertical bars denote S.D. of the mean value of 3–6 separate determinations. The inset shows the percentage of w 3 HxGABA uptake inhibition by Ca2q.
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Fig. 5. Influence of membrane EGTA-washing on the kinetic parameters of the w 3 HxGABA uptake by SPM vesicles. The reactions were carried out for 15 s in the presence of increasing concentrations Ž0.3–10 mM. of w 3 HxGABA. EGTA-washed SPM vesicles Ž`. and native SPM vesicles Žv .. The kinetic parameters values are presented in the inset. The results expressed represent the mean value of 3–6 separate determinations.
Lineweaver–Burk analysis, we calculated Jmax values of 223 pmolrmg protein at 15 s of reaction and K 0.5ŽGABA. of 4 mM for native membranes, whereas these parameters in EGTA-washed membranes were 787 pmolrmg protein and 3 mM, respectively ŽFig. 5, inset.. It appears, therefore, that membrane washing enhances the initial rate of w 3 HxGABA uptake and slightly increases the affinity of the uptake system for GABA. These results indicate that calmodulin removal facilitated the GABA uptake, and that the inhibitory effect of Ca2q was calmodulin-mediated, particularly in the micromolar range of its concentration inside the vesicles. Since in the millimolar range of Ca2q concentration, the Ca2q inhibitory effect on the w 3 HxGABA uptake by SPM was not greatly altered by the calmodulin antagonist, or by EGTA washing of the membranes, it seems that, under these conditions, calmodulin was not the primarily target responsible for the bulk of the Ca2q effect Ž1 mM. as it appears to be the case for the other component of the Ca2q inhibitory effect, observed at micromolar Ca2q concentrations. We also observed that GABA uptake inhibition by 5 mM Ca2q was completely abolished by 1 mM of the phosphatase inhibitor, okadaic acid, and the process was even stimulated by higher concentrations of the drug. In contrast, the inhibitory effect of 1 mM Ca2q was not altered by the phosphatase inhibitor ŽFig. 6.. If we take in consideration that ŽCa Ø calmodulin.-stimulated phosphatase 2B Žcalcineurin. is sensitive to micromolar okadaic acid concentrations w9,49x, it is plausible to assume that it is involved in the Ca2q effect observed. Thus, the inhibition of its activity by 1 mM okadaic acid prevented its response to micromolar concentrations of Ca2q and, therefore, no Ca2q effect was observed under these conditions.
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Fig. 6. Effect of okadaic acid on the w 3 HxGABA uptake inhibition by Ca2q. The assays were performed as described in Fig. 1. The reactions were carried out for 2.5 min in the absence and in the presence of increasing concentrations Ž0.001–10 mM. of okadaic acid, under various experimental conditions Ž2 mM ionomycin, 2 mM ionomycin plus 5 mM CaCl 2 or 2 mM ionomycin plus 1 mM CaCl 2 .. Percentage of inhibition by 5 mM CaCl 2 Ž`. and percentage of inhibition by 1 mM CaCl 2 Žv .. Vertical bars denote S.D. of the mean value of three separate determinations. Inset shows the effect, per se, of okadaic acid on GABA uptake Žabsence of Ca2q and ionomycin..
Conversely, calcineurin appears not be involved in the millimolar Ca2q effect Ž; 60% w 3 HxGABA uptake inhibition by 1 mM Ca2q ., since it remains unchanged at all okadaic acid concentrations studied ŽFig. 6.. On the other
Fig. 7. Effect of calpain inhibitor I on the w 3 HxGABA uptake inhibition by Ca2q. The assays were performed as described in Fig. 1. The reactions were carried out for 2.5 min in the absence and in the presence of 0.5 mM or 5 mM calpain inhibitor I, under various experimental conditions Ž2 mM ionomycin, 2 mM ionomycin plus 5 mM CaCl 2 or 2 mM ionomycin plus 1 mM CaCl 2 .. Percentage of inhibition by 5 mM CaCl 2 Ž`. and percentage of inhibition by 1 mM CaCl 2 Žv .. Vertical bars denote S.D. of the mean value of three separate determinations. Inset shows the effect, per se, of calpain inhibitor I on GABA uptake Žabsence of Ca2q and ionomycin..
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hand, we observed that the inhibitory effect of millimolar Ca2q was partially reduced Ž28%. by the calpain inhibitor I, whereas the micromolar Ca2q effect is even enhanced Ž24%. by the drug ŽFig. 7.. These results were obtained taking in consideration the slight effects that okadaic acid and calpain inhibitor I exert, per se, on the GABA uptake under control conditions Žabsence of Ca2q and ionomycin ŽFig. 6 and Fig. 7, inset.. The results indicate that, at low Ca2q concentrations Žmicromolar range., Ca2q may be a modulator of the GABA transporter activity through the calmodulin-sensitive phosphatase Žcalcineurin., whereas at high concentrations Žmillimolar range., Ca2q may be a non-specific disrupter of the system by reducing membrane potential, by stimulating the proteolytic activity of membrane-bound calpain II and, probably, by altering membrane lipid–protein interactions.
4. Discussion The results reported here show that Ca2q had no significant effect on the GABA uptake by SPM vesicles when it acts at the external side of the vesicles, but it greatly inhibited the process when it was present intravesicularly. Under conditions which do not permit Ca2q entry into the vesicles, the initial velocity of the w 3 HxGABA uptake was not altered in the presence of Ca2q ŽFig. 1A.. Only slight differences occurred with respect to the total amount of w 3 HxGABA accumulated at long periods of reaction ŽFig. 1A.. Indeed, the presence of high Ca2q concentrations in the medium Ž1 mM. caused loss of w 3 HxGABA accumulated by 8 min of reaction, whereas the absence of free Ca2q Žpresence of EGTA. reduced the value of the maximal amount of GABA accumulated which was reached at about 2 min of reaction. Probably, the first effect Žhigh Ca2q concentration. was due to passive diffusion of Ca2q into the vesicles, whereas the second effect Žabsence of free Ca2q . indicates that this cation is useful to maintain the stability of the membrane structure, as previously reported w3,26,30,45x. In contrast to these slight effects promoted by external Ca2q, we observed that, under conditions which permit fast Ca2q entry into the vesicles Žpresence of ionomycin., the GABA uptake by SPM was greatly reduced by Ca2q in a concentration-dependent manner ŽFig. 1.. Interestingly, we found that the Ca2q inhibitory effect is sensitive to the calmodulin antagonists, trifluoperazine and compound 48r80, particularly in the micromolar range of Ca2q concentration ŽFig. 3.. Furthermore, we observed that this sensitivity is only observed when the drugs act intravesicularly. Since we detected Ca2q-dependent interference of calmodulin antagonists with the GABA uptake reaction, we can assume that the Ca2q Ø calmodulin complex is actually involved in the process. In fact, the reduction of the Ca2q effects by these drugs, observed in
the micromolar range of Ca2q concentrations, appears to be mediated by a calmodulin-dependent target involved in the GABA transport process of SPM vesicles. This conclusion is also supported by the results obtained with SPM vesicles washed in an EGTA-containing medium to remove endogenous calmodulin. Under these conditions, the inhibitory effect of micromolar Ca2q on the GABA accumulation process was greatly reduced Ž48%. ŽFig. 4, inset. and we even observed an increment of the maximal amount of GABA accumulated independently of the presence of Ca2q in comparison with the values obtained with native vesicles ŽFig. 4.. Since membrane treatment with EGTA increased the total amount of GABA accumulated in a Ca2q-independent way, it is plausible to assume that, under these conditions, calmodulin removal rather than Ca2q depletion by EGTA was enough to increase GABA uptake. It appears, therefore, that removal or blockage of membrane calmodulin caused liberation of the GABA transporter from a putative Ca2q-dependent, calmodulinmediated mechanism of regulation. This mechanism appears to involve dephosphorylation reactions, since okadaic acid Ž1 mM., which inhibits calcineurin activity w9,47,49x, completely abolished the inhibitory Ca2q effect Žmicromolar range. on the GABA uptake process by SPM vesicles ŽFig. 6.. Indeed, amino acid sequence studies of the GABA transporter molecule showed that it contains several putative phosphorylation sites w1,25x whose function was not yet identified. Moreover, the high sensitivity to Ca2q observed for the target system involved in the GABA transport is in agreement with the high Ca2q sensitivity Ž K 0.5 s 24 nM. of calcineurin w27x, the phosphatase enzyme which is abundant in brain cells where it can be associated to the plasma membrane w2x. It is interesting to note that cyclosporin A, a specific inhibitor of calcineurin w16,32x, was reported to induce stimulation of cloned GABA transporter ŽGAT1. expressed in Xenopus oocytes w12x. These results together with those obtained with phorbol 1-myristate 13-acetate lead the authors to suggest that a modulatory mechanism for the GABA transport Žconversion of the transporter between active and inactive states. may exist at a step subsequent to the protein kinase C-dependent translocation of the GABA transporter to the plasma membrane w12x. It is interesting to note that, in spite of the high hydrophobicity of the drugs used, their incorporation into the vesicle space was necessary to relieve the Ca2q effect on the GABA uptake process. Therefore, it seems that the responsive molecule involved in the process of GABA uptake is preferentially localized at the internal side of the membrane where the lipid soluble drugs accumulated in the hydrophobic core of the membrane w31x are not easily accessible. In contrast to the micromolar component of the inhibitory effect of Ca2q on the GABA uptake by SPM vesicles, the millimolar component of the Ca2q inhibition was not sensitive to these factors, suggesting that, at high
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concentrations, the Ca2q action is mediated by mechanisms other than the activity of the ŽCa P calmodulin.dependent phosphatase, calcineurin. Indeed, this effect appears less specific, reflecting proteolytic cleavage of the GABA transporter by Ca2q-dependent proteases ŽFig. 7., membrane potential variation ŽFig. 2. and, probably, perturbation in the membrane lipid–protein interactions w18– 20,46x. Mechanisms involving ŽNaqrCa2q .-exchanger or phospholipase A 2 activity can not explain the Ca2q effects on the GABA uptake by SPM vesicles, as judged by results obtained with the inhibitors XIP and O ; O-RS-O82, respectively Žresults not shown.. Indeed, 160 mM XIP did not alter the results observed in the presence of intravesicular Ca2q, indicating that the Ca2q effects on the GABA uptake are not related to dissipation of the Naq gradient due to activation of Naq influx in association to Ca2q efflux. Likewise, 400 mM O ; O-RS-O82 did not alter the Ca2q effects observed, suggesting that they are not mediated by arachidonic acid derived from Ca2q activation of phospholipase A 2 which, in spite of its cytosolic localization, could be negligibly associated to the membranes. In the case of glutamate transporter, a relationship between Ca2q inhibition of glutamate release and formation of arachidonic acid has been reported by several investigators. However, these authors used intact retinal cells w15x where the presence of cytosolic phospholipase A 2 may assure production of arachidonic acid. The presence of Ca2q-dependent proteases Žcalpain I and II. in SPM has been previously reported, but they were shown to be easily extracted from the membranes by incubation in a low ionic strength buffer w34,42x. Although our SPM were extensively treated in hypotonic media during the course of their purification, it appears that some of the low Ca2q affinity protease remained associated to the membranes, as judged by the results obtained with the calpain inhibitor I. The results reported here show that Ca2q, in the physiologic range of concentrations, may be a repressor of the GABA transporter activity through a mechanism involving Ca2q Ø calmodulin stimulation of calcineurin. Acknowledgements The authors would like to thank Dr. Kenneth D. Philipson ŽLA, USA. for providing the synthetic peptide inhibitor of the ŽNaqrCa2q .-exchanger, XIP. This research w a s s u p p o rte d b y P R A X IS X X I Ž G ra n t 2r2.1rBIAr224r94.. References w1x S.G. Amara, M.J. Kuhar, Neurotransmitter transporters: Recent progress, Annu. Rev. Neurosci. 16 Ž1993. 73–93.
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w2x D.L. Armstrong, Calcium channel regulation by calcineurin, a Ca2q-activated phosphatase in mammalian brain, Trends Neurosci. 12 Ž1989. 117–122. w3x C. Arias, M. Sitges, R. Tapia, Stimulation of w 3 Hxg-aminobutyric acid release by calcium chelators in synaptosomes, J. Neurochem. 42 Ž1984. 1507–1514. w4x C. Arias, R. Tapia, Differential calcium dependence of g-aminobutyric acid and acetylcholine release in mouse brain synaptosomes, J. Neurochem. 47 Ž1986. 396–404. w5x S. Bernath, Calcium-independent release of amino acid neurotransmitter: Fact or artifact?, Prog. Neurobiol. 38 Ž1992. 57–91. w6x S. Bernath, R.W. Keller, M.J. Zigmond, Release of endogenous GABA can occur through Ca2q-dependent and Ca2q-independent processes, Neurochem. Int. 14 Ž1989. 439–445. w7x S. Bernath, M.J. Zigmond, Characterization of w 3 HxGABA release from striatal slices: Evidence for a calcium-independent process via the GABA uptake system, Neuroscience 27 Ž1988. 563–570. w8x S. Bernath, M.J. Zigmond, Calcium-independent GABA release from striatal slices: The role of calcium channels, Neuroscience 36 Ž1990. 677–682. w9x C. Bialojan, A. Takai, Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics, Biochem. J. 256 Ž1988. 283–290. w10x C.M. Carvalho, C. Bandeira-Duarte, I.L. Ferreira, A.P. Carvalho, Regulation of carrier-mediated and exocytotic release of w 3 HxGABA in rat brain synaptosomes, Neurochem. Res. 16 Ž1991. 763–772. w11x P.H. Chan, R. Kerlan, R.A. Fishman, Intracellular volume of osmotically regulated C-6 glioma cells, J. Neurosci. Res. 8 Ž1982. 67–72. w12x J.L. Corey, N. Davidson, H.A. Lester, N. Brecha, M.W. Quick, Protein kinase C modulates the activity of a cloned g-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter, J. Biol. Chem. 269 Ž1994. 14759–14767. w13x R. Corradetti, G. Moneti, F. Moroni, G. Pepeu, A. Wieraszko, Electrical stimulation of the stratum radiatum increases the release and neosynthesis of aspartate, glutamate and g-aminobutyric acid in rat hippocampal slices, J. Neurochem. 41 Ž1983. 1518–1525. w14x C.B. Duarte, I.L. Ferreira, P.F. Santos, C.R. Oliveira, A.P. Carvalho, Glutamate increases the wCa2q x i but inhibits Ca2q-independent release of w 3 HxGABA in cultured chick retina cells, Brain Res. 611 Ž1993. 130–138. w15x C.B. Duarte, P.F. Santos, J. Sanchez-Prieto, A.P. Carvalho, Gluta´ mate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid, J. Neurosci. Res. 44 Ž1996. 363–373. w16x J. Friedman, I. Weissman, Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA, Cell 66 Ž1991. 799–806. w17x K. Gietzen, Comparison of the calmodulin antagonists compound 48r80 and calmidazolium, Biochem. J. 216 Ž1983. 216–611. w18x D.V. Godin, T.Ng. Wan, Trinitrobenzenesulfonic acid: a possible chemical probe to investigate lipid–protein interactions in biological membranes, Mol. Pharmacol. 8 Ž1972. 426–437. w19x D.V. Godin, T.Ng. Wan, Studies on the membrane-perturbational effects of drugs and divalent cations utilizing trinitrobenzenesulfonic acid, Mol. Pharmacol. 9 Ž1973. 802–819. w20x D.V. Godin, M. Garnett, Perturbational effects of inorganic cations on human erythrocyte membranes, J. Membr. Biol. 28 Ž1976. 143– 168. w21x P.P. Gonc¸alves, M.S. Santos, A.P. Carvalho, Ionic requirements for transport and release of w 3 HxGABA by synaptic plasma membrane vesicles, Neurochem. Int. 17 Ž1990. 401–413. w22x P.P. Gonc¸alves, A.P. Carvalho, Effect of anions on the uptake and release of g-aminobutyric acid by isolated synaptic plasma membranes, Neurochem. Int. 25 Ž1994. 483–492.
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w23x P.P. Gonc¸alves, A.P. Carvalho, Characterization of the carrier-mediated w 3 HxGABA release from isolated synaptic plasma membrane vesicles, Neurochem. Res. 20 Ž1995. 177–186. w24x A.G. Gornall, Ch.J. Bardawill, M.M. David, Determination of serum proteins my means of the biuret reaction, J. Biol. Chem. 177 Ž1949. 751–766. w25x J. Guastella, N. Nelson, H. Nelson, L. Czyzyk, Sh. Keynan, M.C. Miede, N. Davidson, M.A. Lester, B.I. Kanner, Cloning and expression of a rat brain GABA transporter, Science 240 Ž1990. 1303–1306. w26x P. Hess, Calcium channels in vertebrate cells, Annu. Rev. Neurosci. 13 Ž1990. 337–356. w27x L.T. Kakalis, M. Kennedy, R. Sikkink, F. Rusnak, I.M. Armitage, Characterization of the calcium-binding sites of calcineurin B, FEBS Lett. 362 Ž1995. 55–58. w28x N. Kamo, M. Muratsugu, R. Hongoh, Y. Kobatake, Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state, J. Membr. Biol. 49 Ž1979. 105–121. w29x T. Katsu, K. Tasaka, Y. Fujita, Compound 48r80-induced permeability change in liposomal membrane, FEBS Lett. 151 Ž1983. 219–222. w30x R.A. Kauppinen, T.S. Sihra, D.G. Nicholls, Divalent cation modulation of the ionic permeability of the synaptosomal plasma membrane, Biochim. Biophys. Acta 860 Ž1986. 178–184. w31x M.R. Lieber, Y. Lange, R.S. Weinstein, Th.L. Steck, Interaction of chlorpromazine with the human erythrocyte membrane, J. Biol. Chem. 259 Ž1984. 9225–9234. w32x J. Liu, J.D. Farmer Jr., W.S. Lane, J. Friedman, I. Weissman, S.L. Schreider, Calcineurin is a common target of cyclophilin-cyclosporin A and FRBP-FK506 complexes, Cell 66 Ž1991. 807–815. w33x R. Llinas, T.L. McGuiness, C.S. Leornard, M. Sugimori, P. Greengard, Intraterminal injection of synapsin I or calciumrcalmodulindependent protein kinase II alters neurotransmitter release at the squid giant synapse, Proc. Natl. Acad. Sci. USA 82 Ž1985. 3035– 3039. w34x M.N. Malik, M.D. Fenko, H.M. Wisniewski, Purification and partial characterization of two forms of Ca2q-activated neutral protease from calf brain synaptosomes and spinal cord, Neurochem. Res. 9 Ž1984. 233–240. w35x R.A. Nichols, W.C.-S. Wu, J.W. Haycock, P. Greengard, Introduction of impermeant molecules into synaptosomes using freezerthaw permeabilization, J. Neurochem. 52 Ž1989. 521–529. w36x H. Nishio, K. Nezasa, Y. Nakata, Role of calcium ion in platelet serotonin uptake regulation, Eur. J. Pharmacol. 288 Ž1995. 149–155.
w37x T. Noto, H. Hashimoto, J. Nakao, H. Kamimira, T. Nakaijima, Spontaneous release of g-aminobutyric acid formed from putrescine and its enhanced Ca2q-dependent release by Kq stimulation in the brains of freely moving rats, J. Neurochem. 46 Ž1986. 1877–1880. w38x J.-P. Pin, J. Bockaert, Multiple voltage-sensitive calcium channels are probably involved in endogenous GABA release from striatal neurones differentiated in primary culture, Naunyn-Schmiedeberg’s Arch. Pharmacol. 336 Ž1986. 190–196. w39x K. Sato, H. Betz, P. Schloss, The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C, FEBS Lett. 375 Ž1995. 99–102. w40x T.S. Sihra, R.A. Nichols, Mechanisms in the regulation of neurotransmitter release from brain nerve terminals: current hypotheses, Neurochem. Res. 18 Ž1993. 47–58. w41x T.S. Sihra, A.C. Nairn, P. Kloppenburg, Zh. Lin, Ch. Pouzat, A role for calcineurin Žprotein phosphatase-2B. in the regulation of glutamate release, Biochem. Biophys. Res. Commun. 212 Ž1995. 609– 616. w42x R. Siman, M. Baudry, G. Lynch, Purification from synaptosomal plasma membranes of calpain I, a thiol protease activated by micromolar calcium concentrations, J. Neurochem. 41 Ž1983. 950–956. w43x M. Sitges, P.R. Dunkley, L.M. Chiu, A role for calciumrcalmodulin kinaseŽs. in the regulation of GABA exocytosis, Neurochem. Res. 20 Ž1995. 245–252. w44x J.C. Szerb, Relationship between Ca2q-dependent and independent release of w 3 HxGABA evoked by high Kq, veratridine or electrical stimulation from rat cortical slices, J. Neurochem. 32 Ž1979. 1565– 1573. w45x R. Tapia, C. Salazar, Chelation of endogenous membrane calcium inhibits g-aminobutyric acid uptake in synaptosomes, J. Neurosci. Res. 24 Ž1989. 293–298. w46x M.G.P. Vale, A.P. Carvalho, Interaction of chemical probes with sarcoplasmic reticulum membranes, Biochim. Biophys. Acta 601 Ž1980. 620–629. w47x Th.W. Vickroy, W.L. Malphurs, M.L. Carriger, Regulation of stimulus-dependent hippocampal acetylcholine release by okadaic acidsensitive phosphoprotein phosphatases, Neurosci. Lett. 311 Ž1991. 200–204. w48x P.C. Waldmeier, P. Wicki, J.J. Feldtrauer, P.A. Baumann, Ca2q-dependent release of endogenous GABA from rat cortical slices from different pools by different stimulation conditions, NaunynSchmiedebergs Arch. Pharmacol. 339 Ž1989. 200–207. w49x S. Wera, B.A. Hemmings, Serinerthreonine protein phosphatases, Biochem. J. 311 Ž1995. 17–29.
Research report
Regulation of w g- 3 Hxaminobutyric acid transport by Ca2q in isolated synaptic plasma membrane vesicles Paula P. Gonc¸alves b
a,)
, Arselio ´ P. Carvalho b, M. Grac¸a P. Vale
b
a Departamento de Biologia, UniÕersidade de AÕeiro, 3810 AÕeiro, Portugal Centro de Neurociencias, Departamento de Zoologia, Faculdade de Ciencias, UniÕersidade de Coimbra, Coimbra, Portugal ˆ ˆ
Accepted 25 June 1997
Abstract We studied the effect of Ca2q on the transport of the g-aminobutyric acid ŽGABA. by synaptic plasma membrane ŽSPM. vesicles isolated from sheep brain cortex and observed that intravesicular Ca2q inhibits the w 3 HxGABA accumulation in a concentration-dependent manner. This inhibitory effect of Ca2q exhibited two distinct components: one in the micromolar range of Ca2q concentration, and the other in the millimolar range. Previous EGTA washing of the membranes, or incorporation of trifluoperazine into the vesicular space reduced the inhibitory action of Ca2q, particularly at low Ca2q Ž1–5 mM.. Okadaic acid Ž1 mM. also relieved the Ca2q inhibition at low, but not at high Ca2q concentrations Ž1 mM., whereas the calpain inhibitor I did not alter the effect of the low Ca2q, but it partially reduced Ž; 28%. the effect of Ca2q in the millimolar range. The results indicate that the GABA transporter is regulated by low Ca2q concentration ŽmM. and probably its effect is mediated by the ŽCa2q Ø calmodulin.-stimulated phosphatase 2B Žcalcineurin.. In contrast, the GABA uptake inhibition observed at high Ca2q concentrations Ž1 mM. is less specific, and probably it is partially related to the proteolytic activity of membrane bound calpain II. q 1997 Elsevier Science B.V. Keywords: GABA transport; Synaptic plasma membrane; Ca2q regulation
1. Introduction In neuronal synapses, release of g-aminobutyric acid ŽGABA. has been described either by Ca2q-induced exocytosis or by Ca2q-independent reversal of the carrier-mediated transport system at the level of the plasma membrane w40x. We observed previously that the Ca2q-independent process of GABA release by isolated synaptic plasma membrane ŽSPM. vesicles in fact is inhibited by Ca2q under conditions of Kq-induced depolarization, which indicates that Ca2q may modulate the activity of the GABA transporter w23x. The inhibitory action of Ca2q on the carrier-mediated release of neurotransmitters is also supported by the fact that decrease of extracellular Ca2q enhances neurotransmitter release induced by Kq-depolari-
Abbreviations: w 3 HxGABA, w g- 3 Hxaminobutyric acid; Mes, 2-Ž Nmorpholino.-ethanesulfonic acid; SPM, synaptic plasma membraneŽs.. ) Corresponding author. Fax: q351 Ž34. 26-408. 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 2 3 - 4
zation w10,14,37,38x and electrically evoked release w7,13,48x. Furthermore, in platelets, intracellular Ca2q was reported to regulate the uptake of 5-hydroxytryptamine w36x and, in intact retinal cells, Ca2q was shown to inhibit the glutamate transporter by a process which involves production of arachidonic acid due to Ca2q activation of phospholipase A 2 w15x. On the other hand, the results of Tapia and co-workers w45x showed that neutralization of negative surface charges by extracellular Ca2q is necessary for GABA uptake by synaptosomes. It appears, therefore, that Ca2q compartmentation is important for its effects at the level of the neurotransmitter transporters. In spite of these findings, the mechanism involved in the Ca2q regulation of the neurotransmitter transporters remains to be clarified. In presynaptic membranes, phosphorylationrdephosphorylation has been reported to alter the efficiency of neurotransmitter release w33,41,43x. The possibility of GABA transport modulation by second messengers has been considered since 1990, when Guastella et al. w25x, by cloning the cDNA for the rat brain GABA transporter and by expressing the protein sequence in
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oocytes, observed that this protein has up to one putative protein kinase A and seven putative protein kinase C phosphorylation sites. Indeed, it was reported that activators and inhibitors of protein kinase C and protein phosphatases regulate the activity of a cloned rat brain GABA transporter expressed in Xenopus oocytes w12x. More recently, it was demonstrated that this transporter expressed in human embryonic kidney 293 cells is downregulated by protein kinase C activation w39x. In this work, we studied the effect of Ca2q on the GABA transport by SPM vesicles, and the mechanism whereby Ca2q modulates the transporter. We used isolated plasma membranes in our study since this simple vesicle system allows easy changes in the ionic gradients across its membranes and GABA transport machinery can be studied in the absence of exocytotic release of GABA w23x. Our results show that Ca2q regulates the GABA transporter when it works in the forward direction ŽGABA uptake. and this effect is modulated by calmodulin in a process which appears to involve calcineurin.
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2.3. Incorporation of Ca 2 q and calmodulin antagonists into synaptic plasma membrane Õesicles The effect of intravesicular Ca2q on the process of GABA accumulation by SPM vesicles was studied by carrying out reactions of w 3 HxGABA uptake in the presence of 2 mM ionomycin which permits Ca2q entry. On the other hand, the calmodulin antagonists, trifluoperazine and compound 48r80, were incorporated into the vesicles by the freezing and thawing method w35x. Thus, SPM vesicles Ž20 mg proteinrml. were frozen and thawed in the presence of 800 mM trifluoperazine or 780 mgrml compound 48r80 before the initiation of the GABA uptake reaction. After dilution of the membranes in the reaction mixture, the final concentrations of the drugs were about 40 mM and 29 mgrml, respectively. Ionomycin was suitable for Ca2q incorporation into the vesicles, since, in contrast to the freeze–thaw method, it permits Ca2q entry at the time of reaction, avoiding activation of Ca2q proteases due to a delayed contact with Ca2q before starting the GABA uptake reaction.
2. Materials and methods
2.4. Measurement of [ 3 H]GABA uptake by SPM Õesicles
2.1. Materials
The w 3 HxGABA uptake was assayed, at 308C, in a reaction mixture containing 150 mM NaCl, 10 mM HEPES-Na ŽpH 7.4., GABAq w 3 HxGABA Ž0.5 mM, 0.25 mCirml., and 5 mM or 1 mM Ca2q in the presence or absence of ionomycin. The reactions were initiated by addition of the Mes-potassium-loaded vesicles to a final protein concentration of 0.5 mgrml. At several time intervals, the reactions were stopped by rapid filtration of 0.5 ml through glass-fiber filters ŽWhatman GFrB. prewashed with 5 ml of 150 mM NaCl. After filtration of the samples, the filters were washed with 10 ml of the same NaCl solution and the amount of w 3 HxGABA taken up was determined by liquid scintillation spectrometry. The radioactivity on the filters was measured in 8 ml of scintillation cocktail Žcomposition per litter of toluene: 7.3 g of 2,5-diphenyloxazole ŽPPO.; 176 mg of p-bis 2X-Žphenyloxazolyl.benzene ŽPOPOP. and 250 ml of Triton X-100., utilizing a Packard Tri-carb 460 CD liquid scintillation counter. In some assays, the reaction medium contained 2.5 mM valinomycin, 20–100 mM trifluoperazine, 39–80 mgrml compound 48r80, 0.001–10 mM okadaic acid or 0.5–5 mM calpain inhibitor I. Some experiments were carried out with SPM vesicles previously incorporated with trifluoperazine or compound 48r80 as described above.
The 4-amino-n-w2,3- 3 Hxbutyric acid Žw 3 HxGABA., with a specific activity of 92.0 Cirmmol, was obtained from Amersham International. Valinomycin and compound 48r80 were purchased from Sigma-Aldrich, whereas trifluoperazine was supplied by SmithKline and French. The ionomycin and calpain inhibitor I were obtained from Calbiochem-Novabiochem, whereas okadaic acid was supplied by Research Biochemicals International. All reagents were analytical grade. The filters used for SPM retention were obtained from Whatman Co.. 2.2. Isolation of synaptic plasma membrane Õesicles The SPM vesicles were isolated from sheep brain cortex as previously described w21x. After isolation, the vesicles were loaded with Kq by incubating 30 min, at 308C, in 150 mM Mes-potassium salt ŽpH 6.5. and 0.1 mM MgSO4 , at a protein concentration of 5 mgrml. They were then centrifuged for 30 min at 35 000 = g and resuspended in the same buffer to a final protein concentration of about 20 mgrml. The analysis of protein was performed by the method of Gornall w24x and the material was divided into aliquots which were rapidly frozen in liquid nitrogen and stored at y708C. When required, the SPM vesicles were thawed at room temperature. In some experiments, SPM vesicles were washed with 0.1 mM EGTA to remove membrane bound calmodulin. After centrifugation, the trapped EGTA in the pellets was eliminated during the washing in the Kq-loading medium as described above.
2.5. Measurement of the membrane electric potential The Dc of the vesicles were determined at 308C by using a tetraphenyl phosphonium ŽTPPq. selective electrode in a medium containing 8 mM TPPq and 1 mg of
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protein. The Dc of the SPM vesicles was calculated from the accumulation of TPPq according to the following equation w28x:
Dc s 2.3 RTrF log Ž ÕrV . y 2.3 RTrF log Ž 10 F r D E r2.3RT y 1 . The external volume Ž V . is equal to 1 ml and the internal average volume Ž Õ . of SPM vesicles is 3.1 " 0.7 mlrmg protein as determined by the method of Chan et al. w11x. The measured Dc were corrected considering that the Dc of the vesicles is zero when 2.5 mM valinomycin and 150 mM KCl are present in the external medium.
3. Results 3.1. Effect of Ca 2 q on the GABA uptake by synaptic plasma membrane Õesicles The effects of Ca2q on the GABA uptake by SPM vesicles were investigated either when Ca2q was present at the external side of the membrane or when it was localized intravesicularly. Fig. 1A shows the time course of w 3 HxGABA uptake by SPM vesicles under different conditions of Ca2q concentration and Ca2q compartmentation. We observed that extravesicular Ca2q has no significant effect on the process of w 3 HxGABA uptake by SPM vesicles which maximally accumulated 125 pmol GABArmg protein at 2.5 min of reaction. A slight decrease Ž; 12%. was observed in the presence of 0.5 mM EGTA which removes the Ca2q bound to the external side of the membranes. In contrast, in the presence of ionomycin, when Ca2q entered the
vesicles, it inhibited the w 3 HxGABA uptake. The w 3 HxGABA accumulation was maximal Ž99.4 pmol w 3 HxGABA taken uprmg protein. in the presence of EGTA, which indicates that even contaminating Ca2q Žabsence of EGTA. was sufficient to reduce Ž; 22%. the SPM w 3 HxGABA uptake Ž77.5 pmolrmg protein.. In studies of w 3 HxGABA uptake by SPM as a function of Ca2q concentration ŽFig. 1B., we observed that, in the presence of 2 mM ionomycin, the process exhibits two distinct phases: in the micromolar range, Ca2q had a small but significant inhibitory effect on the w 3 HxGABA uptake Ž; 30%. ŽFig. 1B., whereas in the millimolar range Žabove 0.5 mM., w 3 HxGABA accumulation sharply declined, indicating that, under these conditions, intravesicular Ca2q had a strong inhibitory effect, which reached about 70% at 2 mM Ca2q. Control experiments performed in the absence of ionomycin showed no effect on w 3 HxGABA uptake ŽFig. 1B.. Since membrane depolarization induces w 3 HxGABA release w4–8,22,23,44x, we tested whether, under our experimental conditions, membrane potential variations which could be associated with the Ca2q entry into the vesicles would cause inhibition of w 3 HxGABA accumulation. Fig. 2 shows that in a valinomycin-containing medium, where the membrane potential was determined essentially by the potassium ion gradient, the Ca2q inhibitory effects persisted. Indeed, in the presence of ionomycin either contaminant Ca2q Ž; 1.5 mM. in the medium, or 1 mM Ca2q reduced the amount of w 3 HxGABA taken up by the vesicles. It is evident that in the presence of valinomycin, both components of the Ca2q inhibition remained, although the absolute values for w 3 HxGABA accumulated were higher than those observed in the absence of the Kq ionophore.
Fig. 1. Effect of Ca2q on the w 3 HxGABA uptake by SPM vesicles. The SPM vesicles Ž0.5 mg proteinrml. were incubated at 308C in a solution containing 150 mM NaCl, 10 mM HEPES-Na ŽpH 7.4. and 0.5 mM w 3 HxGABA Žsodium-medium. under various experimental conditions. A: time course of w 3 HxGABA uptake in the presence or in the absence of ionomycin. The reactions were carried out in the absence Ž`. and in the presence of 1 mM CaCl 2 Žv ., 0.5 mM EGTA Ž^., 0.5 mM EGTA plus 2 mM ionomycin Ž'., 2 mM ionomycin ŽI., 5 mM CaCl 2 plus 2 mM ionomycin ŽB. or 1 mM CaCl 2 plus 2 mM ionomycin Žl.. The reactions were stopped by filtering 0.5 ml aliquots and the filters radioactivity was measured as described in Section 2. B: w 3 HxGABA uptake as a function of Ca2q concentration. The reactions were carried out for 2.5 min in the absence Ž`. and in the presence of 2 mM ionomycin Žv .. Vertical bars denote S.D. of the mean value of 5–8 separate determinations.
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Fig. 2. Effect of valinomycin on the inhibition of w 3 HxGABA uptake by Ca2q. The reactions were carried out as described in Fig. 1, in the absence ŽA. or in the presence ŽB. of 2.5 mM valinomycin, so that the membrane potential was essentially determined by the Kq gradient. Sodium-medium Ž`.; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA Žv .; sodium-medium plus 2 mM ionomycin Ž^. and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Ž'.. Inset shows the Dc of the SPM vesicles in the absence and in the presence of 2.5 mM valinomycin. Vertical bars denote S.D. of the mean value of 3–5 separate determinations.
This suggests that the electric potential of the membrane is important to determine the extent of the w 3 HxGABA transport but the inhibitory effect of Ca2q persisted independently of the membrane potential magnitude Žy38.6 " 0.7 mV or y25.3 " 1.3 mV in the presence or absence of valinomycin, respectively ŽFig. 2, inset... However, the effect of high Ca2q concentrations ŽmM. was reduced by
about 17% in the presence of valinomycin, whereas that of low Ca2q concentrations ŽmM. was slightly altered Ž5%.. 3.2. Mechanisms inÕolÕed in the Ca 2 q effects on the GABA uptake by synaptic plasma membrane Õesicles We observed that the magnitude of the Ca2q inhibitory effects was reduced when calmodulin antagonists, trifluop-
Fig. 3. Ca2q inhibition of w 3 HxGABA uptake by SPM vesicles containing calmodulin antagonists. The assays were performed as described in Fig. 1. For incorporation of the drug into the vesicles, the SPM vesicles Ž10 mg proteinrml. were freeze–thawed in the presence of 800 mM trifluoperazine or 780 mgrml compound 48r80 before starting the w 3 HxGABA uptake reaction. The final concentrations of trifluoperazine and compound 48r80 in the reaction medium were reduced to 40 mM and 29 mgrml, respectively. The reactions were carried out for 2.5 min under the various experimental conditions. Histograms show from left to right: sodium-medium Žwhite bar.; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA; sodium-medium plus 2 mM ionomycin; sodium-medium plus 2 mM ionomycin and 5 mM CaCl 2 ; and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Žblack bar.. Vertical bars denote S.D. of the mean value of 3–6 separate determinations. The inset shows the percentage of w 3 HxGABA uptake inhibition by Ca2q.
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erazine or compound 48r80, were incorporated into the vesicles ŽFig. 3.. This was particularly evident for the inhibition observed in the micromolar range of Ca2q concentration. The results show that the inhibitory effect of micromolar Ca2q concentrations was 68% reduced by trifluoperazine and completely eliminated by compound 48r80, whereas the inhibitory effect of millimolar Ca2q concentrations was only reduced by 16% or 34% in the presence of intravesicular trifluoperazine or compound 48r80, respectively ŽFig. 3.. Under the experimental conditions used, the trifluoperazine, per se, did not reduce the GABA uptake process. Instead, experiments performed at increasing trifluoperazine concentrations showed that incorporation of the drug into the vesicles promoted a slight enhancement of the w 3 HxGABA accumulation Žresults not shown.. In control experiments, we observed that the simple addition of this drug to the reaction medium did not alter significantly the normal w 3 HxGABA uptake process. Furthermore, the inhibitory effect of intravesicular Ca2q ŽmM or mM. on the w 3 HxGABA uptake was not significantly altered when trifluoperazine was only present outside the vesicles Žresults not shown.. Since similar results of Ca2q effects reduction were obtained with the other calmodulin antagonist, compound 48r80 ŽFig. 3., it appears that a calmodulin-dependent mechanism, localized at the internal side of the membrane, is responsible for the Ca2q inactivation of the GABA transporter in sheep brain SPM vesicles. Compound 48r80 antagonizes the calmodulin function with great specificity w17x. However, its utilization in SPM studies requires appropriate controls since the drug, by increasing membrane permeability to Kq w29x, probably depolarizes the membrane during the
phase of incorporation and, per se, reduces the GABA uptake. Conversely, when it acts extravesicularly, it increases GABA uptake probably by inducing membrane hyperpolarization associated to rapid Kq efflux Žresults not shown.. The observation that the inhibitory effect of micromolar Ca2q was partially relieved by calmodulin antagonists incorporated inside the vesicles strongly suggests that the Ca2q effect may be calmodulin mediated. We further tested this idea by studying the Ca2q effects on the GABA uptake by SPM vesicles previously washed with 0.1 mM EGTA to deplete them of intrinsic calmodulin. We observed that, as in native membranes, Ca2q inhibited the GABA uptake by EGTA-washed vesicles when it entered the vesicles in the presence of ionomycin ŽFig. 4.. It is of interest to note that the inhibitory effect of micromolar Ca2q concentrations was reduced by about 48% in EGTA-washed membranes, whereas the inhibitory effect of millimolar Ca2q concentrations was reduced by only about 7%, as compared to the inhibitory effects of intravesicular Ca2q in native membranes ŽFig. 4, inset.. Furthermore, we also observed that membrane EGTA washing increased the total amount of w 3 HxGABA accumulated either in the absence Ž179 pmolrmg protein per 2.5 min., or in the presence Ž135 pmolrmg protein per 2.5 min. of ionomycin, as compared to the values Ž120 pmolrmg protein per 2.5 min and 78 pmolrmg protein per 2.5 min. obtained for the native membranes under the same experimental conditions, respectively ŽFig. 4.. The results of kinetic studies, depicted in Fig. 5, showed that EGTA washing of the membranes altered the values of Jmax and K 0.5 for the w 3 HxGABA uptake reaction. By
Fig. 4. Comparison between the Ca2q effects on the w 3 HxGABA uptake by native and EGTA-washed SPM vesicles. The reactions were carried out for 2.5 min under the various experimental conditions. Histograms show from left to right: sodium-medium Žwhite bar.; sodium-medium plus 1 mM CaCl 2 ; sodium-medium plus 2 mM ionomycin and 0.5 mM EGTA; sodium-medium plus 2 mM ionomycin; sodium-medium plus 2 mM ionomycin and 5 mM CaCl 2 ; and sodium-medium plus 2 mM ionomycin and 1 mM CaCl 2 Žblack bar.. Vertical bars denote S.D. of the mean value of 3–6 separate determinations. The inset shows the percentage of w 3 HxGABA uptake inhibition by Ca2q.
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Fig. 5. Influence of membrane EGTA-washing on the kinetic parameters of the w 3 HxGABA uptake by SPM vesicles. The reactions were carried out for 15 s in the presence of increasing concentrations Ž0.3–10 mM. of w 3 HxGABA. EGTA-washed SPM vesicles Ž`. and native SPM vesicles Žv .. The kinetic parameters values are presented in the inset. The results expressed represent the mean value of 3–6 separate determinations.
Lineweaver–Burk analysis, we calculated Jmax values of 223 pmolrmg protein at 15 s of reaction and K 0.5ŽGABA. of 4 mM for native membranes, whereas these parameters in EGTA-washed membranes were 787 pmolrmg protein and 3 mM, respectively ŽFig. 5, inset.. It appears, therefore, that membrane washing enhances the initial rate of w 3 HxGABA uptake and slightly increases the affinity of the uptake system for GABA. These results indicate that calmodulin removal facilitated the GABA uptake, and that the inhibitory effect of Ca2q was calmodulin-mediated, particularly in the micromolar range of its concentration inside the vesicles. Since in the millimolar range of Ca2q concentration, the Ca2q inhibitory effect on the w 3 HxGABA uptake by SPM was not greatly altered by the calmodulin antagonist, or by EGTA washing of the membranes, it seems that, under these conditions, calmodulin was not the primarily target responsible for the bulk of the Ca2q effect Ž1 mM. as it appears to be the case for the other component of the Ca2q inhibitory effect, observed at micromolar Ca2q concentrations. We also observed that GABA uptake inhibition by 5 mM Ca2q was completely abolished by 1 mM of the phosphatase inhibitor, okadaic acid, and the process was even stimulated by higher concentrations of the drug. In contrast, the inhibitory effect of 1 mM Ca2q was not altered by the phosphatase inhibitor ŽFig. 6.. If we take in consideration that ŽCa Ø calmodulin.-stimulated phosphatase 2B Žcalcineurin. is sensitive to micromolar okadaic acid concentrations w9,49x, it is plausible to assume that it is involved in the Ca2q effect observed. Thus, the inhibition of its activity by 1 mM okadaic acid prevented its response to micromolar concentrations of Ca2q and, therefore, no Ca2q effect was observed under these conditions.
111
Fig. 6. Effect of okadaic acid on the w 3 HxGABA uptake inhibition by Ca2q. The assays were performed as described in Fig. 1. The reactions were carried out for 2.5 min in the absence and in the presence of increasing concentrations Ž0.001–10 mM. of okadaic acid, under various experimental conditions Ž2 mM ionomycin, 2 mM ionomycin plus 5 mM CaCl 2 or 2 mM ionomycin plus 1 mM CaCl 2 .. Percentage of inhibition by 5 mM CaCl 2 Ž`. and percentage of inhibition by 1 mM CaCl 2 Žv .. Vertical bars denote S.D. of the mean value of three separate determinations. Inset shows the effect, per se, of okadaic acid on GABA uptake Žabsence of Ca2q and ionomycin..
Conversely, calcineurin appears not be involved in the millimolar Ca2q effect Ž; 60% w 3 HxGABA uptake inhibition by 1 mM Ca2q ., since it remains unchanged at all okadaic acid concentrations studied ŽFig. 6.. On the other
Fig. 7. Effect of calpain inhibitor I on the w 3 HxGABA uptake inhibition by Ca2q. The assays were performed as described in Fig. 1. The reactions were carried out for 2.5 min in the absence and in the presence of 0.5 mM or 5 mM calpain inhibitor I, under various experimental conditions Ž2 mM ionomycin, 2 mM ionomycin plus 5 mM CaCl 2 or 2 mM ionomycin plus 1 mM CaCl 2 .. Percentage of inhibition by 5 mM CaCl 2 Ž`. and percentage of inhibition by 1 mM CaCl 2 Žv .. Vertical bars denote S.D. of the mean value of three separate determinations. Inset shows the effect, per se, of calpain inhibitor I on GABA uptake Žabsence of Ca2q and ionomycin..
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hand, we observed that the inhibitory effect of millimolar Ca2q was partially reduced Ž28%. by the calpain inhibitor I, whereas the micromolar Ca2q effect is even enhanced Ž24%. by the drug ŽFig. 7.. These results were obtained taking in consideration the slight effects that okadaic acid and calpain inhibitor I exert, per se, on the GABA uptake under control conditions Žabsence of Ca2q and ionomycin ŽFig. 6 and Fig. 7, inset.. The results indicate that, at low Ca2q concentrations Žmicromolar range., Ca2q may be a modulator of the GABA transporter activity through the calmodulin-sensitive phosphatase Žcalcineurin., whereas at high concentrations Žmillimolar range., Ca2q may be a non-specific disrupter of the system by reducing membrane potential, by stimulating the proteolytic activity of membrane-bound calpain II and, probably, by altering membrane lipid–protein interactions.
4. Discussion The results reported here show that Ca2q had no significant effect on the GABA uptake by SPM vesicles when it acts at the external side of the vesicles, but it greatly inhibited the process when it was present intravesicularly. Under conditions which do not permit Ca2q entry into the vesicles, the initial velocity of the w 3 HxGABA uptake was not altered in the presence of Ca2q ŽFig. 1A.. Only slight differences occurred with respect to the total amount of w 3 HxGABA accumulated at long periods of reaction ŽFig. 1A.. Indeed, the presence of high Ca2q concentrations in the medium Ž1 mM. caused loss of w 3 HxGABA accumulated by 8 min of reaction, whereas the absence of free Ca2q Žpresence of EGTA. reduced the value of the maximal amount of GABA accumulated which was reached at about 2 min of reaction. Probably, the first effect Žhigh Ca2q concentration. was due to passive diffusion of Ca2q into the vesicles, whereas the second effect Žabsence of free Ca2q . indicates that this cation is useful to maintain the stability of the membrane structure, as previously reported w3,26,30,45x. In contrast to these slight effects promoted by external Ca2q, we observed that, under conditions which permit fast Ca2q entry into the vesicles Žpresence of ionomycin., the GABA uptake by SPM was greatly reduced by Ca2q in a concentration-dependent manner ŽFig. 1.. Interestingly, we found that the Ca2q inhibitory effect is sensitive to the calmodulin antagonists, trifluoperazine and compound 48r80, particularly in the micromolar range of Ca2q concentration ŽFig. 3.. Furthermore, we observed that this sensitivity is only observed when the drugs act intravesicularly. Since we detected Ca2q-dependent interference of calmodulin antagonists with the GABA uptake reaction, we can assume that the Ca2q Ø calmodulin complex is actually involved in the process. In fact, the reduction of the Ca2q effects by these drugs, observed in
the micromolar range of Ca2q concentrations, appears to be mediated by a calmodulin-dependent target involved in the GABA transport process of SPM vesicles. This conclusion is also supported by the results obtained with SPM vesicles washed in an EGTA-containing medium to remove endogenous calmodulin. Under these conditions, the inhibitory effect of micromolar Ca2q on the GABA accumulation process was greatly reduced Ž48%. ŽFig. 4, inset. and we even observed an increment of the maximal amount of GABA accumulated independently of the presence of Ca2q in comparison with the values obtained with native vesicles ŽFig. 4.. Since membrane treatment with EGTA increased the total amount of GABA accumulated in a Ca2q-independent way, it is plausible to assume that, under these conditions, calmodulin removal rather than Ca2q depletion by EGTA was enough to increase GABA uptake. It appears, therefore, that removal or blockage of membrane calmodulin caused liberation of the GABA transporter from a putative Ca2q-dependent, calmodulinmediated mechanism of regulation. This mechanism appears to involve dephosphorylation reactions, since okadaic acid Ž1 mM., which inhibits calcineurin activity w9,47,49x, completely abolished the inhibitory Ca2q effect Žmicromolar range. on the GABA uptake process by SPM vesicles ŽFig. 6.. Indeed, amino acid sequence studies of the GABA transporter molecule showed that it contains several putative phosphorylation sites w1,25x whose function was not yet identified. Moreover, the high sensitivity to Ca2q observed for the target system involved in the GABA transport is in agreement with the high Ca2q sensitivity Ž K 0.5 s 24 nM. of calcineurin w27x, the phosphatase enzyme which is abundant in brain cells where it can be associated to the plasma membrane w2x. It is interesting to note that cyclosporin A, a specific inhibitor of calcineurin w16,32x, was reported to induce stimulation of cloned GABA transporter ŽGAT1. expressed in Xenopus oocytes w12x. These results together with those obtained with phorbol 1-myristate 13-acetate lead the authors to suggest that a modulatory mechanism for the GABA transport Žconversion of the transporter between active and inactive states. may exist at a step subsequent to the protein kinase C-dependent translocation of the GABA transporter to the plasma membrane w12x. It is interesting to note that, in spite of the high hydrophobicity of the drugs used, their incorporation into the vesicle space was necessary to relieve the Ca2q effect on the GABA uptake process. Therefore, it seems that the responsive molecule involved in the process of GABA uptake is preferentially localized at the internal side of the membrane where the lipid soluble drugs accumulated in the hydrophobic core of the membrane w31x are not easily accessible. In contrast to the micromolar component of the inhibitory effect of Ca2q on the GABA uptake by SPM vesicles, the millimolar component of the Ca2q inhibition was not sensitive to these factors, suggesting that, at high
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concentrations, the Ca2q action is mediated by mechanisms other than the activity of the ŽCa P calmodulin.dependent phosphatase, calcineurin. Indeed, this effect appears less specific, reflecting proteolytic cleavage of the GABA transporter by Ca2q-dependent proteases ŽFig. 7., membrane potential variation ŽFig. 2. and, probably, perturbation in the membrane lipid–protein interactions w18– 20,46x. Mechanisms involving ŽNaqrCa2q .-exchanger or phospholipase A 2 activity can not explain the Ca2q effects on the GABA uptake by SPM vesicles, as judged by results obtained with the inhibitors XIP and O ; O-RS-O82, respectively Žresults not shown.. Indeed, 160 mM XIP did not alter the results observed in the presence of intravesicular Ca2q, indicating that the Ca2q effects on the GABA uptake are not related to dissipation of the Naq gradient due to activation of Naq influx in association to Ca2q efflux. Likewise, 400 mM O ; O-RS-O82 did not alter the Ca2q effects observed, suggesting that they are not mediated by arachidonic acid derived from Ca2q activation of phospholipase A 2 which, in spite of its cytosolic localization, could be negligibly associated to the membranes. In the case of glutamate transporter, a relationship between Ca2q inhibition of glutamate release and formation of arachidonic acid has been reported by several investigators. However, these authors used intact retinal cells w15x where the presence of cytosolic phospholipase A 2 may assure production of arachidonic acid. The presence of Ca2q-dependent proteases Žcalpain I and II. in SPM has been previously reported, but they were shown to be easily extracted from the membranes by incubation in a low ionic strength buffer w34,42x. Although our SPM were extensively treated in hypotonic media during the course of their purification, it appears that some of the low Ca2q affinity protease remained associated to the membranes, as judged by the results obtained with the calpain inhibitor I. The results reported here show that Ca2q, in the physiologic range of concentrations, may be a repressor of the GABA transporter activity through a mechanism involving Ca2q Ø calmodulin stimulation of calcineurin. Acknowledgements The authors would like to thank Dr. Kenneth D. Philipson ŽLA, USA. for providing the synthetic peptide inhibitor of the ŽNaqrCa2q .-exchanger, XIP. This research w a s s u p p o rte d b y P R A X IS X X I Ž G ra n t 2r2.1rBIAr224r94.. References w1x S.G. Amara, M.J. Kuhar, Neurotransmitter transporters: Recent progress, Annu. Rev. Neurosci. 16 Ž1993. 73–93.
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