Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Journal of Neurochemistry Raven Press, Ltd., New York 0 199 1 International Society for Neurochemistry

Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response

of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Paulino Gbmez-Puertas, Albert0 Martinez-Serrano, Pablo Blanco, Jorgina Satdstegui, and Elena Bogbnez

Departamento de Biologia Molecular, Centro de Biologia Molecular, CSIC- Universidad Autdnoma de Madrid, Madrid, Spain

Abstract: Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High-K+ depolarization in media of low

(32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K+-stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na+-based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 pM veratridine at 0.5 mM external calcium.

Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) ofthe phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed. Key Words: Synaptosomes-K+ depolarization-Calcium uptake-Protein dephosphorylation- Transient dephosphorylation-Acetylcholine release. Gbmez- Puertas P. et al. Conditions restricting depolarization-dependent calcium influx in synaptosomes reveal a graded response of P96 dephosphorylation and a transient dephosphorylation of P65. J. Neurochem. 56, 2039-2047 (199 1).

Depolarization-induced calcium influx into nerve terminals through voltage-sensitive calcium channels (VSCC) is generally accepted as the trigger for neurotransmitter release (Katz, 197 1). However, the molecular mechanisms underlying the coupling between the increase in cytosolic free calcium concentration ( [Ca2'Ii) and release remain unknown. One hypothesis to explain how calcium ions could exert their triggering action proposes that calcium-dependent phosphorylation of proteins of the nerve terminal initiates synaptic vesicle movement and exocytosis (Burke and De Lorenzo, 1982; Nestler and Greengard, 1982). During the past few years, direct evidence for a role of phosphorylation of specific proteins in the regulation of neurotransmitter release has been obtained (Llinhs et al., 1985; Dekker et al., 1989; Nichols et al., 1990).

In contrast to the numerous studies dealing with the role of protein phosphorylation in the process of neurotransmitter release, reports concerning the physiological function of protein dephosphorylation induced by increases in the intracellular calcium concentration are sparse. However, it has been shown that in some cellular systems dephosphorylation of a specific phosphoprotein could be one of the events controlling exocytosis. Thus, synchronous exocytosis in Paramecium tetraurelia involves the rapid and reversible dephosphorylation of a 63-65-kDa phosphoprotein (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985) present in a wide variety of species and tissues (Satir et al., 1989).

In synaptosomes, depolarization by high external Kf results in the calcium-dependent phosphorylation of

Received August 15, 1990; revised manuscript received November 12, 1990; accepted December 4, 1990.

Address correspondence and reprint requests to Dr. E. Bogbnez at Departamento de Biologia Molecular, Centro de Biologia Molecu- lar, CSIC-Universidad Autcinoma de Madrid, 28049 Madrid, Spain.

Abbrevialions used: ACh, acetylcholine; Arg', arginine; BSA, bovine serum albumin; Cho+, choline; [Ca2+],, cytosolic free calcium concentration; fura-2/AM, fura-2 acetoxymethyl ester; SDS, sodium dodecyl sulfate; VSCC, voltage-sensitive calcium channel.

2039

2040 P. GOMEZ-PUERTAS ET AL.

many proteins, as well as a decrease in the phosphorylation level of several phosphoproteins (Krueger et al., 1977; Robinson and Dunkley, 1983, 1985; Wang et al., 1988). The synaptosomal protein undergoing dephosphorylation that has received much attention is a phosphoprotein of 96 kDa (P96). Dephosphorylation of P96 is apparently regulated not simply by an increase in the cytosolic calcium concentration, but also by the mechanism of calcium entry (through calcium channels). Thus, P96 shows a rapid dephosphorylation (within 5 s) only when the increase in [Ca2+ji is induced by depolarization of the synaptosomal plasma membrane (Robinson and Dunkley, 1985; Robinson et al., 1987).

We are interested in P96 dephosphorylation because we found in previous work that the reduction in calcium channel-mediated 45Ca2+ uptake observed in synaptosomes from aged rats is correlated with a slower dephosphorylation of P96 (Martinez-Serrano et al., 1989). We reasoned that if P96 senses the calcium influx through VSCC, the reduced dephosphorylation of P96 could result from the decreased activity of VSCC in aged animals. At present, little is known about how the temporal course of calcium influx is related to the rate of P96 dephosphorylation. Therefore, the main goal of this study was to investigate temporal changes in phosphorylation level of synaptosomal phosphoproteins, in particular P96, in response to high-K+ depolarization under conditions in which calcium entry through VSCC could be greatly reduced, i.e., depolarization by high K+ at external pH below physiological pH (Nachshen and Blaustein, 1979; Umbach, 1982; Prod'hom et al., 1987) at submillimolar concentrations of external calcium.

MATERIALS AND METHODS

Materials Fura-2 acetoxymethyl ester (fura-2/AM) was obtained from

Molecular Probes (Eugene, OR, U.S.A.); Ficoll400 was from Pharmacia (Uppsala, Sweden); veratrine [assumed to consist of 25% (wt/wt) veratridine], acetylcholinesterase (EC 3.1,1.7), choline oxidase (EC 1.1.3.17), and peroxidase (EC 1.1 1.1.7) were from Sigma (St. Louis, MO, U.S.A.); and 5-amino- 1,2,3,4-tetrahydrophthalazinedione (luminol) was from Merck (Darmstadt, F.R.G.). 45Ca2+ and "Pi were from Amersham (Bucks, U.K.). All other chemicals were the high- est grade available.

Preparation of synaptosomes Synaptosomes from whole rat brain were prepared ac-

cording to the method of Booth and Clark (1978), as described previously (Vit6rica and Satnistegui, 1986), using a discon- tinuous Ficoll gradient.

Protein phosphorylation Synaptosomes ( 5 mg/ml) were prelabeled with 32Pi (0.5

mCi/ml) for 30 rnin at 37°C under an oxygen atmosphere in basal medium containing 5 mM KCI, 10 mM glucose, 1 mM MgClz, 0.5 mM CaC12, 10 mM Tris-HC1, and either 145 mM NaCl (Na+-based medium) or 113 mM choline (Cho+)-HCI plus 32 mM NaCl (Cho+-based medium) at pH

7.4 or 6.5, respectively. After 32Pi prelabeling, synaptosomes were incubated for the times indicated in the figures in an equal volume of basal or high-K+ medium (with no calcium added, so that the final concentration of CaC12 under depolarization was 0.25 mM). The tonicity of the high-K+ media was maintained by decreasing the NaCl or Cho+-HC1 concentrations in the Na+- or Cho+-based media, respectively (62 mM K+ final concentration). During veratridine depolarization, synaptosomes prelabeled with 32Pi in Na+-based medium were incubated with an equal volume of the same medium containing 40 pM veratridine. Incubations were ter- minated by addition of a sodium dodecyl sulfate (SDS) solution, and proteins were separated by SDS-polyacrylamide electrophoresis in 7.5- 15% linear gradient gels, as previously described (Martinez-Serrano et al., 1989). Quantitation of protein phosphorylation on autoradiograms was performed using a 300 A Computing densitometer (Molecular Dy- namics).

45Ca2+ uptake Synaptosomes ( 5 mg/ml) were preincubated for 5 min at

30°C in Na+- or Cho+-based media at pH 7.4 or 6.5, respectively. After preincubation, synaptosomes were transferred to basal or high-K+ media, and 45Ca2+ accumulation was measured at 30°C for various time periods by a filtration technique, essentially as described previously (Vitbrica and Sathstegui, 1986).

[Ca2+li measurement [Ca"], was determined using fura-2/AM, as previously de-

scribed (Martinez et al., 1988; Martinez-Serrano and Saths- tegui, 1989). Briefly, synaptosomes (10 mg/ml) were loaded with the probe by incubation in Na+- or Cho+-based media at pH 7.4 or 6.5, respectively, that contained 1% (wt/vol) bovine serum albumin (BSA) and 5 pM fura-2/AM for 30 rnin at 37°C. After the loading period, synaptosomes were washed twice to eliminate unhydrolyzed esters, resuspended (10 mg/ml) in basal media that contained 0.1% (wt/vol) BSA, and stored in the dark at room temperature for no more than 2 h. Fluorescence was determined at 37°C with wavelengths of 340 nm for excitation (2-mm window) and 505 nm for emission (2-mm window) using a Schoeffel RRlOOO spectro- fluorometer with temperature-controlled cuvette and mag- netically driven stirrer. Data were digitized and stored in a Hewlett Packard HP86B microprocessor. Records, expressed in terms of [Ca2+Ii, were obtained by averaging independent experiments.

Acetylcholine (ACh) release ACh release was measured using the chemiluminescence

method of Israel and Lesbats (198 1) modified for neutral pH. Synaptosomes (2 mg/ml) were preincubated for 30 rnin at 37°C in Na+-based medium (pH 7.4) or Arg+-based medium (pH 6.5, with the same composition as Cho+-based medium except for the substitution of Cho+-HC1 by Arg+-HCI) containing 10 U/ml acetylcholinesterase, 10 U/ml choline oxidase, 1 1 U/ml peroxidase, and 40 pM luminol. After preincubation, synaptosomes were placed in a LKB 1251 lumi- nometer to measure continuous release of ACh. Depolarization assays were performed by injecting a concen- trated solution of KCl to give a final concentration of 38 mM. Luminescence responses evoked by K+ were quantified by comparison with the luminescence output of ACh standards added directly to the cuvette following K+ stimulation. Endogenous content of ACh before K+ stimulation was de-

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DEPHOSPHOR YLA TION AND CALCIUM INFLUX 204 1

termined as previously described (Willoughby et al., 1986). Synaptosomes were preincubated in 1 ml of basal medium for 30 min at 37°C at a protein concentration of 1 mg/ml. Then, trichloroacetic acid [20% (wt/vol) final concentration] was added and the samples were processed for ACh determination (Willoughby et al., 1986). Aliquots (100 PI) were assayed with 900 pl of chemiluminescence buffer cocktail ( 1 36 mM NaCl, 56 mM KCl, 1.2 mM MgClz, 1 mM CaClZ, 10 mM Tris-HC1 at pH 8.6, 10 U of acetylcholinesterase, 10 U of choline oxidase, 1 1 U of peroxidase, and 40 pA4luminol). To measure the endogenous content of Cho+ and its release from K+-stimulated synaptosomes, acetylcholinesterase was omitted from the incubation mixtures and 100 pM physostigmine was added to inhibit endogenous acetylcholinesterase.

Other methods ATP concentration was determined as described by Le-

masters and Hackenbrock (1978) using an ATP biolumines- cence test (Boehringer-Mannheim, F.R.G.). Protein content was determined by the biuret method. Means of different groups of data were compared using Student's t tests.

RESULTS

Protein phosphorylation under depolarization conditions that restrict calcium entry

To investigate the response of synaptosomal phosphorylation and dephosphorylation systems to high- K+ depolarization under conditions restricting calcium entry, we have studied the pattern of changes in protein phosphorylation when synaptosomes are incubated in media of low Na+ concentration (32 mM) that was held constant through the depolarization pulse (Cho+- based media). Under this condition of depolarization, the contribution of the [Ca2'],/[Na']i exchange to the entry of calcium into synaptosomes is minimal and calcium influx proceeds mainly through VSCC (Cou- tinho et al., 1984; Martinez et al., 1987). Moreover, to limit calcium entry through VSCC, we used 0.25 mM CaC12 and an external pH of 6.5.

Prelabeling of synaptosomes in Cho+-based medium did not alter the general phosphorylation pattern com-

FIG. 1. Autoradiograrn showing the effect of the incubation media and K+ depolarization on endogenous protein phosphorylation. Synap- tosomes were prelabeled with 32Pi as described in Materials and Methods in Naf-based media (A) or Cho+-based media (6). Aliquots were transferred to basal or high-K+ media and incubated for the indicated times. Arrows point to the phosphoproteins P96, synapsin I (SPI), P65, and P42.

pared with Na+-based medium, with two exceptions- the phosphoproteins of apparent molecular masses 65 and 42 kDa (P65 and P42, Robinson and Dunkley, 1983). Basal level of phosphorylation of P65 increased in Cho+-based medium, whereas P42 showed the op- posite behaviour (Fig. 1).

When synaptosomes prelabeled with 32Pi in Cho+- based medium were depolarized with 62 mM K+, the temporal changes in protein phosphorylation were markedly different compared with depolarization in Na+-based medium (Fig. 1). Quantitative analysis of the increases in phosphorylation of a representative phosphoprotein, synapsin I, showed that in Na+-based medium the maximal level of phosphorylation was at- tained after approximately 5-10 s of depolarization. However, in Cho+-based medium, phosphorylation of synapsin I increased slowly, the maximal increase being only 8.44 f 1.22% after 30 s of depolarization (Fig. 2A). The possibility that a diminished rate of phosphorylation of synapsin I resulted from differences in the internal pool of ATP was ruled out as the synaptosomal concentration of ATP was not significantly different in Cho+- and Na+-based media (data not shown). A slower rate of change in the state of phosphorylation of P96 was also detected during depolarization in Cho+-based medium (Fig. 2B). P96 was rapidly dephosphorylated in Na'-based medium, as found by Robinson and Dunkley (1985), losing 57.3 +- 2.32% of phosphate content within 5 s of depolarization. In Cho+-based medium, a gradual dephosphorylation of P96 was observed, and a new steady-state level of phosphorylation was reached only after 1 min of potassium addition. Dephosphorylation of phosphoprotein P42, that possibly corresponds to the cy subunit of the mitochondria1 enzyme pyruvate dehydrogenase (Robinson and Dunkley, 1983), was also slower in Cho+- than in Na+-based media, but the differences were significant only during the first 10 s of depolarization (Fig. 2C). The phosphate loss was maximal after a 15-s depolarization pulse, and represented 50.46 k 2.95% of basal phosphate content.

J. Neurochem., Vol. 56. No. 6, 1991


800

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Depolarization in Cho+-based medium not only slowed down temporal increases and decreases in the phosphorylation level of synaptosomal phosphoproteins, but also induced responses not previously observed. P65 is a phosphoprotein that is rapidly labeled during incubation of synaptosomes with '*Pi and that does not show significant changes in phosphorylation in response to depolarization (Robinson and Dunkley, 1983). However, it is surprising that depolarization by high K+ in Cho+-based media induced transient dephosphorylation of P65 that was reversed within 10 s. After that, phosphorylation increased and a new steady- state level of phosphorylation was obtained after 30 s that represented 83.39 * 2.57% of the basal level (Fig. 2D).

FIG. 3. Effect of K+ depolarization on calcium uptake and [Ca2+]i in synaptosomes incubated in Na+- or Cho+-based media. A Synapto- somes were preincubated for 5 min at 30°C in Na+-based media (0) or Cho+-based media (0). Aliquots were then transferred to basal or high-K' media containing 0.25 mM CaCI, (0.75 WCi of .SCa2'/ml), and %a2+ accumulation was measured at the times indicated. Results correspond to K+-stimulated "Ca2+ uptake (uptake in 62 mM K+ media minus uptake in basal, 5 mM media), and are means rt SEM from three experiments performed in triplicate. B: Average [Caz']i records from three independent experiments showing the effect of high-K+ depolarization (62 mM) in synaptosomes loaded with fura-P/AM in Na+- or Cho+-based media, in the presenceof 0.25 mM CaCI, .

FIG. 2. Time-course of K+ depolarization-induced phosphorylation/dephosphorylation of individual phosphoproteins in Na+-based media (0) or Cho+- based media (0). Synaptosomes were prelabeled with 32P, and depolarized as described in the legend to Fig. 1. Phosphorylation in protein bands was quantified by measuring peak areas on densitometric tracings after subtracting background densities under the peak. A Synapsin I (SPI). B: P96. C P42. D: P65. Phosphor- ylation is expressed as a percentage of the value of nondepolarized controls. Results are means f. SEM from four to seven experiments.

Influx of calcium during depolarization in Cho+-based media

To ascertain whether the changes in protein phosphorylation observed in Cho+-based media were in fact correlated with a limited entry of calcium into synaptosomes, and whether this could be paralleled by modifications in [Ca"],, we examined 45Ca2+ uptake and [Ca2+Ii transients following K+ depolarization of synaptosomes in both incubation media. Figure 3A shows the time-course of K+-stimulated 45Ca2+ uptake in Na+- and Cho+-based media. As predicted, depolarization in Cho+-based media resulted in a reduction of calcium influx. The 45Ca2+ uptake value in a 2-s pulse of depolarization, that should more precisely reflect the amount of calcium entering through VSCC,

- A B

TIME ( 5 ) T I M E (min)

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DEPHOSPHOR YLA TION AND CALCIUM INFLUX 2043

was 83% lower than that observed in Na+-based media. When [Ca2+Ii was measured by fura-2 fluorescence under resting conditions (Fig. 3B), the [Ca2+Ii in synaptosomes incubated in Cho+-based media was slightly but reproducibly lower (365 versus 427 nM). Depo- larization of synaptosomes in Na+- or Cho+-based media led to markedly different changes in [Ca2+Ii. De- polarization of synaptosomes in Na+-based media gave the characteristic peak [Ca2+Ii that immediately de- clined to a plateau level in agreement with previous observations using fura-2 or quin-2 (Ashley et al., 1984; Hansford and Castro, 1985; Adam-Vizi and Ashley, 1987; Komulainen and Bondy, 1987). In contrast, depolarization of synaptosomes in Cho+-based media caused a severe reduction in both peak and plateau of [Ca2+Ii. It could be argued that extracellular acidification at low [Na+Io resulted in a drop in internal pH, as shown by Nachshen and Drapeau (1988), and therefore the differences in [Ca2+Ii could reflect changes in KD for Ca2+-fura-2. We do not think this interpretation is likely for the following reasons: (a) the internal pH in synaptosomes preincubated in Cho+-based media may drop from the physiological value of approximately 7.0 (Nachshen and Drapeau, 1988) to a value close to that of the external medium, i.e., 6.5; and (b) as reported by Drapeau and Nachshen (1988), the KD for Ca2+-fura-2 does not change significantly in the pH range 6.2-7.0. Therefore, the values of [Ca2+Ii that we have found in Cho+-based media under resting and depolarizing conditions are not underestimated, but represent real values.

K+-evoked ACh release Similar to K+-dependent calcium uptake, K+-evoked

neurotransmitter release from synaptosomes exhibits a fast phase, which terminates within the initial few seconds of depolarization (Nachshen and Blaustein, 1980; Drapeau and Blaustein, 1983; Floor, 1983; Susz- kiw and OLeary, 1983; Leslie et al., 1985). It is thought that the fast phase corresponds to the activation of VSCC that are linked to the release process at active zones. To investigate whether the reduction in K+- stimulated 45Ca2+ uptake and [Ca2+Ii transients observed in Cho+-based media is paralleled by a decrease in neurotransmitter release, we measured the release of endogenous ACh in response to K+ depolarization. Because Cho+ present in our media interfered with the determination of ACh, we replaced Cho+ with Arg' as a countercation. This substitution did not modify the results of the phosphorylation and calcium uptake studies (data not shown).

Figure 4A illustrates the typical responses of luminescence output when ACh release was evoked by KCl injection in Na+- or Arg+-based media. K+-evoked ACh release was dependent on extracellular calcium during both depolarization conditions, as the addition of 2 mM EGTA resulted in 80% reduction of release (not shown). When 100 pM physostigmine was present during preincubation and depolarization to inhibit

acetylcholinesterase, light emission was not detected after KC1 injection, as expected if only ACh and not Cho+ is released by depolarization of synaptosomes (Israel and Lesbats, 198 1; Willoughby et al., 1986). ACh release from synaptosomes depolarized in Arg+-based media was markedly reduced compared with that found in Na+-based media, as determined from the maximal amplitude and slope of the luminescence peak (Fig. 4B). It is unlikely that these results are due to differences in the endogenous content of ACh, or to an effect of pH on the ACh detection system because the endogenous content of ACh was the same in the two synaptosomal preparations at the end of the preincubation period (105 pmol/mg of protein), and because standard solutions of ACh gave identical responses in both incubation media (Fig. 4A).

Protein phosphorylation under depolarization by veratridine

The preceding studies indicated that during depolarization of synaptosomes under conditions in which

n . - I \

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FIG. 4. K+-evoked ACh release. A Representative luminescence tracings of the ACh release evoked by K+ depolarization in synaptosomes incubated in Na+- or Arg+-based media. Synaptosomes were preincubated and assayed for ACh release as described in Materials and Methods. Following each luminescence response due to ACh release, calibration standards (1 5 and 20 pmol) were injected. B: Comparison of the maximal amplitude and slope of the luminescence signal produced by K+ depolarization in Na+- or Arg+-based media. Results are means k SEM from four independent experiments performed in duplicate.

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calcium influx through VSCC is restricted, both protein phosphorylation/dephosphorylation rates and evoked release of ACh were greatly reduced. However, because a specific effect of an associated change in internal pH could not be excluded, we decided to examine the effect of a different depolarizing agent, known to slow down calcium uptake, ACh release, and [Ca2+Ii transients in synaptosomes, on the temporal changes in protein phosphorylation. Veratridine depolarizes plasma membrane by activation of voltage-dependent Na+ channels (Blaustein and Goldring, 1 975). Depolariza- tion of synaptosomes by veratridine causes a slower increase in [Ca2+Ii than that obtained with K+ depolarization, and no rapid peak elevation occurs (Rich- ards et al., 1984; Hansford and Castro, 1985; Adam- Vizi and Ashley, 1987). Moreover, "Ca2+ uptake and [ I4C]ACh release are also reduced following veratridine depolarization (Adam-Vizi and Ligeti, 1986; Adam- Vizi and Ashley, 1987).

When 32Pi-prelabeled synaptosomes were depolarized by 20 pM veratridine in the presence of 0.5 mM CaC12, the temporal changes in phosphorylation of synapsin I were significantly modified with respect to those observed during K+ depolarization in Na+-based media. Phosphorylation was slower, and the maximal phosphorylation level was markedly reduced (Fig. 5A). Similar to the results obtained in Cho+-based media, dephosphorylation of P96 was slower, and the steady level of phosphorylation was reached only after 1 min of veratridine addition (Fig. 5B). In contrast, the time- courses of P42 dephosphorylation were not significantly different during veratridine and K+ depolarization (Fig.

5C). It is interesting that a transient dephosphorylation of P65 was also observed when synaptosomes were depolarized by veratridine (Fig. 5D), which closely re- sembles the pattern obtained during K+ depolarization in Cho+-based media (compare Fig. 2D with Fig. 5D).

DISCUSSION

In the present work, temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization under conditions restricting calcium influx have been studied. Synaptosomes preincubated in Cho+-based media maintain a polarized plasma membrane, as the membrane potential is affected neither by partial substitution of Na' by Cho+ (Martinez et al., 1987) nor by acidification of the external medium (Deutsch et al., 1981). Consistent with these observations, synaptosomes preincubated in Cho+-based media at pH 6.5 retained the capacity to regulate [Ca2+]i, although at a level slightly lower than that of synaptosomes preincubated in Na'-based media, possibly due to the well known influence of acidification of the external medium on calcium accumulation in synaptosomes (Snelling and Nicholls, 1985). Synaptosomes also responded to K+ depolarization by taking up calcium, which was reflected in the K+-stimulated uptake of 45Ca2+ and the elevation of [Ca2+Ii. However, partial substitution of and external acidification brought about pronounced effects on the magnitude of these responses. K+-stimulated 45Ca2+ uptake during a 2-s pulse of depolarization was reduced by SO%, as was the K+-induced release of en-

_ _ 0 10 20 30 40 50 60

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FIG. 5. Time-course of protein phosphorylation/dephosphorylation following veratridine depolarization. Synaptosomes were prelabeled with 32PI in Na'-based media and then incubated with 20 juLl veratridine (0) for the indicated times. Quantitation of phosphorylation in phosphoprotein bands was performed as described in the legend to Fig. 2. For comparative purposes, the data from high-K+ depolarization in Na+-based media from Fig. 2 are also included (0). A Synapsin I (SPI). B: P96. C: P42. D: P65. Phosphorylation is expressed as a percentage of the value of nondepolarized controls. Results are means t SEM from four experiments.

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DEPHOSPHOR YLA TION AND CALCIUM IMFL UX 204.5

dogenous ACh, and the peak [Ca2'Ii was barely de- tectable. If, as suggested by Adam-Vizi and Ashley (1 987), the initial fast K+-induced ACh release is related to the initial burst of Ca2' entry and the resultant peak [Ca2+Ii, the reduction in K+-induced ACh release that we observed is consistent with the decrease in initial 45Ca2+ uptake and the severe fall in peak [Ca2'Ii. Under these experimental conditions, the most relevant find- ings were those related to the phosphoproteins P96 and P65.

Modulation of p96 dephosphorylation rate Previous studies in synaptosomes demonstrated a

depolarization-induced decrease in the phosphorylation level of P96 that was dependent on the presence of extracellular calcium (Krueger et al., 1977; Robinson and Dunkley, 1983), and apparently on the mechanism of calcium entry (Robinson and Dunkley, 1985; Rob- inson et al., 1987). The calcium dependency of P96 dephosphorylation was reported to be an all-or-none phenomenon, in contrast to the graded responses to different depolarizing stimuli and degree of plasma membrane depolarization shown by other synaptoso- ma1 phosphoproteins (Robinson and Dunkley, 1983). However, K+-induced dephosphorylation of P96 in synaptosomes from 24-month-old rats is significantly slower than in synaptosomes from 3-month-old rats, the reduction in dephosphorylation rate being correlated with the decrease in calcium uptake via calcium channels that occurs in old animals (Martinez-Serrano et al., 1989). These results suggest that the dephosphorylation rate of P96 could be modulated by the amount of calcium entering the presynaptic terminal. The results we are presenting here indicate that P96 dephosphorylation rate can indeed show a graded response to the rate of calcium uptake under various depolarization conditions. Significant differences in the initial loss of phosphate content following K+ depolarization were found between Cho+-based media and Na+-based media. Moreover, we detected a slower dephosphorylation of P96 on exposure to veratridine, where Ca2+ uptake is also slower than under K' depolarization and thought to proceed mainly through Na+ channels (Adam-Vizi and Ligeti, 1986; Adam-Vizi and Ashley, 1987). This discrepancy between our results and those of Robinson and Dunkley (1983) could be due to the fact that we have examined the response to veratridine depolarization at lower veratridine and external calcium concentrations. In conclusion, our results show that, similar to synapsin I phosphorylation, P96 dephosphorylation could be modulated by the amount of calcium entering the synaptosome. In this context, it is interesting to note that P96 was found to be localized in cytosolic fractions from synaptosomal lysates (Robinson et al., 1987), and we have detected it in crude synaptic vesicles from 32Pi-labeled synaptosomes (manuscript in preparation). Studies are in progress to determine the precise location of this phosphoprotein and whether, as proposed for synapsin I, it

could be involved in the regulation of the process of neurotransmitter release.

Transient dephosphorylation of P65 This is the first time a transient dephosphorylation

of P65 following depolarization is described in synaptosomes. As P65 is very rapidly labeled during incubation of synaptosomes with 32Pi, it was proposed that P65 is located within mitochondria (Robinson and Dunkley, 1983). However, we could not detect any 65- kDa phosphoprotein in isolated mitochondria labeled with either 32Pi or [Y-~~P]ATP. Rapid incorporation of radiolabeled phosphate into P65 could be explained by a high turnover rate of phosphate. P65 became more highly phosphorylated during prelabeling in Cho+- based media than during prelabeling with Na+-based media. This could result from a shift in the relative activities of the protein kinase and protein phosphatase responsible for the phosphorylation/dephosphorylation cycle; i.e., it could be a consequence of changes in the affinity of the protein phosphatase for Ca2+ brought about by acidification of the internal pH. Acidification has substantial effects on the affinity of a variety of enzymes and components of the cytoskeleton for Ca2+ (Fabiato and Fabiato, 1978; El-Saleh and Solaro, 1988; Missiaen et al., 1989).

Transient dephosphorylation of P65 is associated with calcium influx into synaptosomes, as it was not observed when depolarization occurred in the absence of added calcium and in the presence of EGTA. It may not have been detected previously if the process nor- mally takes place in a very short time and is rapidly counterbalanced by rephosphorylation, both processes (dephosphorylation and rephosphorylation) having been slowed down during the depolarization conditions under which we observed them. At present, analysis of temporal changes in phosphorylation in most exocytotic systems can be done only on a time scale of seconds. Thus, transient dephosphorylation of P65 oc- curring in less than 1 s would not have been detected in previous studies.

Calcium entry in synaptosomes in response to K+ depolarization is biphasic (Nachshen and Blaustein, 1980, 1982). The initial rapidly decaying phase of calcium influx is associated with the opening and subse- quent inactivation of calcium channels, which occurs over a period of several hundred milliseconds (Nach- shen, 1985; Suszkiw et al., 1986, 1989). There is evidence suggesting that these channels mediate the phasic release of neurotransmitters from synaptosomes (Dra- peau and Blaustein, 1983; Floor, 1983; Suszkiw and O'Leary, 1983; Leslie et al., 1985). It is interesting that P65 dephosphorylation is a phasic or transient process, as are calcium channel activation and transmitter release.

What could be the physiological significance of P65 dephosphorylation in the presynaptic terminal? Gilli- gan and Satir (1 982) demonstrated that when normal exocytosis is induced in wild-type cells of Paramecium

J. Neurochem., Vol. 56, No. 6, 1991


tetraurelia, rapid dephosphorylation of a 63-kDa phosphoprotein is detected. Dephosphorylation is very rapid (5 1 s), reverses within 20 s, and strictly parallels the actual amount of exocytosed organelles (Zieseniss and Plattner, 1985). It has been reported that this phosphoprotein is involved in the regulation of membrane fusion during exocytosis (Stecher et al., 19871, and that calcineurin or calcineurin-like proteins are also involved (Momayezi et al., 1987). Satir et al. ( 1 989) have demonstrated recently that polyclonal antibodies against the purified phosphoprotein (parafusin) detect a 63-kDa polypeptide in other unicellular organisms and in several metazoan groups, concluding that this molecule may play a fundamental role in the mechanism of exocytosis in most eukaryotic cells. Clearly, it will be important to determine whether the synapto- soma1 phosphoprotein P65 is immunologically similar to parafusin.

Acknowledgment: This work was supported by grants 841 0323 and 8610520 from the Comisi6n Interministerial de Ciencia y Tecnologia and by institutional grants from the Fundaci6n Ram6n Areces and FIS to the Centro de Biologia Molecular. P.G.-P., A.M.-S., and P.B. were recipients of grants from the Ministerio de Educaci6n y Ciencia.

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