Differentiation of the Ca2+-stimulated binding from the Cl−-dependent binding of [3H]glutamate in synaptic membranes from rat brain

Neuroscience Research, 4 (1986) 129-142

129

Elsevier Scientific Publishers Ireland Ltd. NSR 00160

Differentiation of the Ca 2 +-stimulated binding from the C1--dependent binding of [3H]glutamate in synaptic membranes from rat brain Kiyokazu Ogita and Yukio Yoneda Department of Pharmacology, Setsunan University,Hirakata, Osaka 573-01 (Japan) (Received 19 May 1986; Accepted 30 June 1986)

Key words: [3H]Glutamate binding; C1--dependent binding; Ca2÷-stimulated binding; Anion transport carrier; Quisqualic acid; 2-Amino-4-phosphonobutyric acid; Hydrophilic SH-reactive agent; L-Ascorbic acid

SUMMARY The effect of Ca 2 ÷ as well as C1- ions on [3H]glutamate (Glu) binding was re-examined using rat brain synaptic membranes frozen at - 80 °C in 0.32 M sucrose. The inclusion of 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate disclosed the C1- -dependent binding or Ca 2 ÷ stimulated binding even at 2 min after the initiation of incubation at 30 °C and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate. Seatchard analysis revealed that CI - as well as CI -/Ca 2 ÷ ions invariably caused a significant increment of the number of binding sites without altering their affinity, whereas Na ÷ ions induced a prominent increment of the density of binding sites with a concomitant lowering of their affinity. DL-2-Amino-4-phosphonobutyric acid selectively abolished the CI--dependent and Ca ~÷-stimulated bindings without significantly affecting the basal or Na÷-dependent binding. Quisqualic acid induced a profound inhibition of both Cl--dependent and Ca2+-stimulated bindings, to a significantly greater extent than that of the basal and Na÷-dependent bindings. D-Aspartic acid exhibited a potent inhibition of the Na ÷ -dependent binding with a significantly less potent displacement of the basal, C1- -dependent and Ca 2 ÷ -stimulated bindings. An inhibitor of anion transport, 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS), not only eliminated the C1- -dependent binding, but also completely abolished the Ca 2 ÷ -stimulated binding. Scatchard analysis revealed that DIDS (0.1 mM) prevented the C1-- and CI-/Ca 2÷ -induced increment of the density of binding sites with no significant change of their affinity. Pretreatment of the membranes with hydrophilic SH-reaetive agents such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) invariably resulted in a more sensitive inhibition of the Ca 2 ÷ -stimulated binding than that of the C1--dependent binding, while hydrophobic reagent p-chloromercuribenzoic acid produced a similarly potent elimination of the C1--dependent and

Correspondence: Y. Yoneda, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01, Japan. 0168-0t02/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.

130 Ca2~ -stimulated bindings. Calcium-stimulated binding was also found to be sensitively diminished o) dithiothreitol and dithioerythritol as compared with the CI-dependent binding. In vitro addition of L-ascorbic acid (10 6-10 3 M) attenuated the Ca2+-stimulated binding to a significantly greater exten~ than the inhibition of the CI- -dependent binding. These results support the proposal that Ca2 ' ions may elicit a significant potentiation of [3H]Glubinding through interaction with some membranous constituents distinctly different from the CI--dependent binding sites.

INTRODUCTION It has been believed that the high-affinity and N a + -independent binding of radiolabeled L-glutamic acid (Glu) corresponds to the association of this putative excitatory neurotran smitter in the mammalian central nervous system (CN S) 11,~6 with its phy siologically relevant synaptic receptors 22"z8, whereas the Na +-dependent binding represents the N a + -dependent uptake sites of this neuroactive amino acid 15,24.3~. Chloride ions potentiate the N a +-independent binding ~3"2a while inducing a diminution of the N a + -dependent binding 2'17"21. Calcium ions also exhibit an additional enhancement of the C I - - d e p e n d e n t binding in a strictly C1--dependent manner 2t. These biochemical binding studies along with electrophysiological investigations have revealed the multiplicity of the receptors for central acidic amino acid neurotransmitters: N-methylD-aspartic acid (NMDA)-sensitive receptors (A 0, quisqualic acid (QA)-sensitive receptors (A2), kainic acid (KA)-sensitive receptors (m3) and L-2-amino-4-phosphonobutyric acid (AP-4)-sensitive receptors (A4) 18"32. On the other hand, the functional significance as well as the exact molecular mechanisms underlying the C l - - d e p e n d e n t and Ca2+-stimulated bindings are still controversial. For example, the C1 - -dependent binding has been supposed to represent an anion driven uptake of GIu 14"18'27or reflect the linkage of Glu receptors to the anion channels 36. Several lines of evidence have indicated a potential involvement of Ca 2 + dependent proteases 3-5 in the exhibition of the Ca z + -stimulated binding. It has been also shown that freezing of the membrane preparations completely eliminates this Ca 2 + -stimulated binding selectively ~z. These previous findings led us to re-examine the effect of these ions and attempt to differentiate the Ca 2 + -stimulated binding from the C1 - -dependent binding using frozen synaptic membranes at - 80 °C in 0.32 M sucrose in order to clarify the molecular mechanisms underlying the Ca 2 + -induced augmentation of the binding. MATERIALS AND METHODS

Materials [ 3H ]Glu (L-3,4-[ 3H ]glutamic acid, 44.1 Ci/mmol) was purchased from New England Nuclear (Boston, MA, U.S.A.) and purified by column chromatography on Dowex 50W × 8 (200-400mesh) resin. QA, N M D A , KA, DL-AP-4, L-glutamic acid diethylester (GDEE), p-chloromercuribenzoic acid (PCMB), 5,5'-dithiobis-(2-nitro-

131 benzoic acid) (DTNB) and L-ascorbic acid were obtained from Sigma Chemicals (St. Louis, Mo, U.S.A..) N-Ethylmaleimide (NEM) was purchased from Nakarai Chemicals (Osaka, Japan). Other chemicals used were all of commercially guaranteed grade.

Membrane preparation Crude synaptic membrane fractions were obtained from whole brains of adult male albino Wistar rats weighing 200-250 g according to the procedures described by Zukin et al.39 with a minor modification33,34. In brief, the brains were homogenized in 15 vols. of 0.32 M sucrose using a Teflon-glass homogenizer at 4 °C following the sacrifice of animals by decapitation. The crude synaptic membrane fractions were suspended in 50 vols. of 50 mM Tris-acetate buffer (pH 7.4) using a Polytron homogenizer at setting no. 6 for 30 s and the suspensions were centrifuged at 50,000 x g for 30 min at 4 °C. After repeating these washing procedures by 50 mM Tris-acetate buffer (pH 7.4) 3 times, the final pellets were suspended in 0.32 M sucrose and the suspensions were frozen at - 80 ° C until required for use 3s. The frozen suspensions were thawed at room temperature and washed twice by the buffer as above before each use. Under these storage conditions, no significant alteration was detected in the basal, CI--dependent, Ca 2 +-stimulated and Na ÷ -dependent bindings of [3H]GIu for at least 1 month. Binding assay The final pellets were resuspended in the buffer and the suspensions were incubated with 10 nM [3H]Glu in 500 #1 50 mM Tris-acetate buffer (pH 7.4) at 30 °C for 60 min unless indicated otherwise. Incubation was terminated by the addition of 3 ml ice-cold buffer and subsequent filtration on a Whatman GF/B glass filter under a constant vacuum of 15 mm Hg. After washing the filter 4 times with 3 ml cold buffer, the radioactivity trapped on the filter was measured by a liquid scintillation spectrometer (LSC 900, Aloka, Japan) using 5 ml modified Triton-toluene scintillant 3s at a counting efficiency of 40-42%. The radioactivity found in the presence of 1 mM non-radioactive Glu was subtracted from each experimental value to obtain the specific binding in accordance with the y-aminobutyric acid (GABA) receptor binding assay system3°'35. Chloride-dependent binding was calculated by subtraction of the basal binding from the binding in the presence of 20 mM ammonium chloride. The latter binding was also subtracted from the binding obtained in the presence of both 20 mM ammonium chloride and 2.5 mM calcium acetate to estimate the Ca2+-stimulated binding. The Na + -dependent binding was obtained from the difference between the basal binding and the binding found in the presence of 100 mM sodium acetate. For examining the effect of SH-reactive agents on the binding, synaptic membranous suspensions (2.5 mg protein/ml) were preincubated with varying concentrations of the reagents in 2.5 ml of 50 mM Tris-acetate buffer (pH 7.4) at 30 °C for 30 min. Preincubation was terminated by the addition of 5.5 ml chilled buffer and subsequent centrifugation at 50,000 g for 30 min at 4 °C. The resultant pellets were suspended in the same buffer and the suspensions were again centrifuged as above. The final pellets

132 thus obtained were resuspended in the buffer and the suspensions were subjected to the routine binding assay. For Scatchard analysis, suspensions were incubated with 10 or 20 nM [3H]Glu at 30 °C for 60 min in the presence of varying concentrations of non-radioactive Glu over a concentration range from 10 to 1190 nM. Binding parameters such as Ko and B ..... were calculated from the slope and intercept of the straight line drawn by a computer with a non-linear regression analysis programme, respectively. Binding assay was carried out in the triplicate with a variation of less than 109/o. Protein content was determined by the method of Lowry et al. 2°. Results were usually expressed as the mean + S.E.M. and the statistical significance was determined by Student's t-test.

RESULTS

Characterization of the binding sites Scatchard analysis revealed that [3H]GIu binding sites consisted of a single component with a K,~ of 0.51 + 0.07/~M and a Bmax of 4.30 + 0.79 pmol/mg protein, respectively, rather than being comprised of two separate dements. The basal binding reached a plateau within 30 min of incubation of 30 °C (Fig. 1). The inclusion of C1 or C I - / C a 2÷ ions showed the C1--dependent binding or Ca 2÷ -stimulated binding, respectively, even at 2 min after the initiation of the incubation and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate (Fig 1). Calcium as well as chloride ions caused a significant increment of the

"~800 ~6oo

72~oc m

N

~t) ~ - ~ 2 ~ ~2 0 mM N.I--t4CI '

,~.....~ ~

~5

? r en

I

10

I

20

I

I

II

!

30 40 60 Time (rain) Fig. 1. The effect of incubation time on [3H]glutamate (Glu) binding. Membrane suspensions were incubated with 10 nm [3H]Gluin 50 mM Tris-acetate buffer(pH 7.4) containing either 20 mM ammonium chloride, 20 mM ammoniumchloride plus 2.5 mM calcium acetate or 100 mM sodium acetate at 30 °C for varying periods. Each value represents the mean _+S.E.M. obtained from 3 separate experiments.

133 density of binding sites without altering their affinity (20 mM NH4C1, Kd = 0.56 + 0.05, B m a x = 16.6 + 1.13; 2 0 m M NH4C1 + 2.5mM Ca (CH3COO)2, K a = 0.69 + 0.06, B ~ x = 28.8 + 2.87), whereas sodium ions produced a remarkable lowering of the affinity with a concomitant increment of the density (100 mM CHaCOONa, K a = 1.17 + 0.12, B m a x = 43.2 + 2.5). L-Glu exhibited a concentration dependent disA o L-GLU • D-GLU a L-ASP • L-CSA

o 100

g

~I00

--q__i

g 0

"6 x3 .--50 (:0

"6

?

i15

5o o L-GDEE a=QUIKA

""\~

E ra D I

0

~-7 -6

-5

-4

0

-3 Log M

B

1oo .~

~ Basal [-" *

~

CI-Depcndcnt Ca-Stimulated

10 -s M

10 -~ M GDEE

10 .3 M

Fig. 2. The effect of various neuroactive compounds on [3H]glutamate (Glu) binding. Crude synaptic membranes were incubated with 10 nM [3H]GIu in 50 mM Tris-aeetate buffer (pH 7.4) containing varying concentrations of the compound indicated at 30 °C for 60 rain in the absence (A) and presence (B) of either 20 mM ammonium chloride, 20 mM ammonium chloride plus 2.5 mM calcium acetate or 100 mM sodium acetate. Chloride-dependent binding was calculated by subtracting the basal binding from the binding found in the presence of 20 mM ammonium chloride. The latter binding was also subtracted from the binding found in the presence of both 20 mM ammonium chloride plus 2.5 mM calcium acetate to obtain the Ca 2+stimulated binding. Sodium-dependent binding was estimated from the difference between the basal binding and the binding found in the presence of 100 mM sodium acetate. Each value represents the mean + S.E.M. obtained from 3 separate experiments. * P < 0.05, compared with each value for the basal binding. L-ASP, L-aspartic acid; L-CSA, L-cysteinesulfinic acid; L-GDEE, L-glutamic acid diethylester; L-GLU, L-glutamic acid; D-GLU, D-glutamic acid; KA, kainic acid; NMDA, N-methyl-D-aspartic acid; QUI, quisqualic acid.

134 placement of the basal [3H]Glu binding. L-Aspartic acid (L-Asp) as well as L-cysteinesulfinic acid (L-CSA) induced a similarly potent inhibition of the binding, while D-Glu elicited less potent inhibition. D-Asp had no significant inhibitory effect on the binding ( 1 0 - T M , 110+12.5~o; 1 0 - 6 M , 1 0 7 . 0 _ 0 . 6 ; 1 0 - S M , 103.0_+3.8; 1 0 - 4 M , 125.0 _ 13.5). The binding was also potently inhibited by QA, an agonist for certain subclasses of the central Glu receptors, but not by agonists for other subclasses such as N M D A and KA. An antagonist for the QA-sensitive receptors, L-GDEE, exerted a moderately potent attenuation of the binding (Fig. 2A). The latter antagonist induced a greater inhibition of the Ca e+-stimulated binding than that of other 3 different bindings (Fig. 2B). To elucidate the functional significance of the individual binding sites, the effect of L- and D-Asp was examined. D-Asp is well known to have a high affinity similar to L-Asp for the Na ÷ -dependent sites of Glu uptake with a considerably lower affinity for the Glu receptors than that of L-Asp 16. As shown in Table I, L-Asp exhibited a profound inhibition of the basal binding, whereas D-Asp had no significant action on the basal binding. L-Asp also elicited a prominent displacement of the bindings found in the presence of 20 mM ammonium chloride and 20 mM ammonium chloride plus 2.5 mM calcium acetate to a significantly greater extent than that by D-Asp. D-Asp as well as L-Asp, however, exerted a similarly potent inhibition of the binding found in the presence of 100 mM sodium acetate (Table I). These results suggest that the specific binding of [3H]Glu to synaptic membranes in the presence of Na ÷ ions represents the association of this putative neurotransmitter with its uptake sites. DL-AP-4 induced a potent suppression of the C1 - -dependent and Ca 2 ÷ -stimulated bindings with no significant change in the basal and Na ÷ -dependent bindings (Fig. 3A). QA exhibited a similarly potent inhibition of the C1--dependent and Ca 2 ÷ -stimulated bindings to a significantly greater extent than that of the basal binding, while inducing a considerably less potent inhibition of the Na ÷-dependent binding (Fig.3B). Other agonists such as N M D A and KA had no such significant effect on the binding. The data described above all support the assumption that Ca2+-stimulated as well as

TABLE I THE EFFECT OF L- AND D-ASPARTATEON [3H]GLUTAMATEBINDING Addition

None 20 mM NH4CI 20 mM NH4Cl + 2.5 mM Ca (CH3COO)2 100 mM CH3COONa * P < 0.05, compared with the value for L-aspartate.

IC5o (#M) L-Aspartate

D-Aspartate

6.1 _ 0.5 19.3 + 0.7 18.3 _+4.4 2.8 _ 0.4

No inhibition 120 _+12.0" 88.3 + 7.3* 3.2 _+ 0.4

135 A ~.~HzCI

+2.5 mM Ca(CH3COO)2

i: 400 100 mM CH3COONa ~ '

i

200 03 t~

E

None

~.

~

• *

D T

~,

0

o

-6'

-5'

DL-AP4 (Log M)

-;,

-;

B [ ] Basal [ ] Cl- Oel~ndcnt [ ] Ca-Stimulated • Na-Depcndent

U

"15 o

10-5

10-4

lO-a

Q A (M)

rh .,.,

i m•"r

0 lo-S

10-4

NMDA(M)

10- 3

,z.

/I/1/ 10-5

10-a

10-3

K A(M)

Fig. 3. The effect of an antagonist (A) and agonists (B) for central glutamate (Gin) receptors on [SH]Glu binding. Synaptic membranous preparations were incubated with 10 nM [3H]GIu in 50 mM Tris-acetate buffer (pH 7.4) containing either 20 mM ammonium chloride, 20 mM ammonium chloride plus 2.5 mM calcium acetate or 100 mM sodium acetate at 30 °C for 60 men in the presence of varying concentrations of DL-2-amino-4-phosphonobutyric acid (AP4), quisqualic acid (QA), N-methyl-D-aspartic acid (NMDA) or kainic acid (KA). Each value represents the mean + S.E.M. obtained from 3-4 independent experiments. * P < 0.05, ** P < 0.01, compared with each control value.

CI--dependent bindings may demonstrate pharmacological characteristics similar to the central Glu receptors rather than those of Glu uptake sites in terms of the susceptibility to various neuroactive compounds including D-Asp, DL-Ap-4 and QA. In vitro addition of an anion transport inhibitor 4,4'-diisothiocyanatostilbene2,2'-disulfonic acid (DIDS) 1"29not only resulted in a prevention of the Cl--induced increment of the density of binding sites, but also prevented the additional increment of the density by Ca2 + ions (Table II). These results suggest that Ca2 + ions may elicit

136 TABLE II THE EFFECT OF 4,4'-DIISOTHIOCYANATOSTILBENE-2,2'-DISULFONIC ACID (DIDS) ON [3H]GLUTAMATE BINDING Addition

K d (#M)

B.... (pmol/mg protein)

20 20 20 20

0,56 0,41 0.69 0,78

16.6 +_ 1.1 3.65 _+0.56* 28.8 + 2.9 5.19 +_0.97*

mM mM mM mM

NH4CI NHnCI + 0.1 mM DIDS NH4C1 + 2.5 mM Ca (CH3COO)2 NHnC1 + 2.5 mM Ca (CH3COO)2 + 0.1 mM DIDS

+_0.07 _+0.05 + 0.06 + 0.15

* P < 0.01, compared with each control value.

their additionally stimulatory action on the C1 - -dependent binding through interacting with the DIDS-sensitive anion transport carriers.

Effect of SH-reagents Since the t'mdings cited above all supported the similarity between C1 and Ca 2 ~ ions in the molecular mechanisms underlying the enhancement of binding, the effect of the chemical modification of the membranous constituents was examined in order to differentiate the latter two bindings. Membrane suspensions were pretreated with Basal tO0

u

~

50

10-5M

[]

10-z' M

•

10-3M

0

.=-

PCMB

E

h~

"~

[]

NEM

DTNB

C[-Dependent

50

*

*

0

PCMB

NEM

Ca-Stimulated

,

DTNB

50

*

PCMB

NEM

DTNB

Fig. 4. The effect of pretreatment with SH-reactive agents on [3H]glutamate (Glu) binding. Synaptic membranes (2.5 mg protein/ml) were preincubated with varying concentrations of SH-reactive agents such as p-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM) or 5,5'-dithiobis-(2-nitrobenzoicacid) (DTNB) in 2.5 ml 50 mM Tris-acetate buffer (pH 7,4) at 30 °C for 30min. This preincubation was terminated by the addition of 5.5 ml ice-cold buffer and subsequent centrifugation at 50,000 g for 30 min at 4 °C. Following the repetition of these washing procedures twice, the final pellets were resuspended in the buffer and the suspensions were incubated with I0 nM [3H]GIu in 50 mM Tris-acetate buffer (pH 7.4) at 30 ° C for 60 min in the presence of either 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate. Each value represents the mean + S.E.M. obtained from 3 separate experiments. * P < 0.05, ** P < 0.01, compared with each control value.

137 Basal

DTT

DTE

[]

10"s M

[]

lo-~ u

•

lo -3 M

2-ME

Ca-Stirr~a~ed

o - Oependerd ~I00

:

:

o lmr/m O

DTT

DTE

:fLml

100

.:%::::

2-ME

::~ : : : :

DTT

:::::

::::

DTE

:::::;::: : ::

2-ME

Fig. 5. The effect of various SH-containing compounds on [3H]glutamate (Glu) binding. Membranous suspensions were incubated with 10 nM [3H]Gluin 50 mM Tris-acetate buffer(pH 7.4) containing either 20 mM ammoniumchlorideor 20 mM ammoniumchlorideplus 2.5 mM calciumacetate at 30 °C for 60 min in the presence of varying concentrations of SH-containing compounds such as dithiothreitol (DTT), dithioerythritol(DTE) and 2-mercaptoethanol(2-ME). Each value represents the mean + S.E.M. obtained from 3 independent experiments. * P < 0.05, compared with each control value. varying concentrations of SH-reactive agents such as PCMB, N E M and DTNB in Tris-acetate buffer at 30 °C for 30 min. After washing the membranes with the buffer twice, the binding of [3H]Glu was determined in the presence of either 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate (Fig. 4). It was found that each SH-reactive agent invariably induced a significant and concentration-dependent inhibition of the basal, CI--dependent and Ca 2 ÷ -stimulated bindings. In particular, Ca 2 ÷-stimulated binding was most sensitively abolished by pretreatment with N E M (Fig. 4). Pretreatment with 1 0 - 4 M N E M resulted in the complete abolishment of the Ca 2 ÷ -stimulated binding whereas it reduced the basal and C1--dependent bindings by less than 5 0 ~ . In vitro addition of SH-containing compounds such as dithiothreitol (DTT) and dithioerythritol (DTE) also yielded a selective inhibition of the Ca 2 ÷ -stimulated binding to a significantly greater extent than that of the basal and Cl--dependent bindings (Fig. 5). These results suggest that membranous SH-residues may scarcely be responsible for the exhibition of the selective diminution of the Ca 2 ÷-stimulated binding by SH-reactive agents as well as SH-containing compounds. To study such a possibility, the effect of L-ascorbic acid, a reducing agent without SH-moiety, was examined. As shown in Fig. 6, ascorbate elicited a complete abolishment of the Ca 2 ÷-stimulated binding with a concurrent inhibition of the C1--dependent binding by 5 5 ~ even at a considerably low concentration of 10-3 M. The inclusion of higher concentrations of ascorbate resulted in a concentration-dependent elimination of the basal and C1-dependent bindings. These results clearly indicate that L-ascorbic acid quite selectively abolishes the Ca 2 ÷ -stimulated component of [3H]GIu binding in synaptic membrane preparations.

138 o

E]]]] Basal

t

~J

"5

G1oo

~ - * * -]

÷

~

CI-Dependent

¢-

"o ¢-

¢,n

E

5 ? "1-

10 -6

10 -5

10 -4

10 -3

L - A s c o r b i c Acid ( M )

Fig. 6. The effect of L-ascorbic acid on [3H]glutamate (Gin) binding. Synaptic membrane suspensions were incubated with 10 nM [3H]GIu in 50 mM Tris-acetate buffer (pH 7.4) containing either 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate at 30 °C for 60 min in the presence of varying concentrations of ascorbic acid. Each value represents the mean + S.E.M. obtained from 4 separate experiments. * P < 0.05, ** P < 0.01, compared with the value for the basal binding.

DISCUSSION

In this study, it has been demonstrated that C a 2+ ions elicit an additionally stimulatory action on the C1--induced augmentation of the binding of [3H]GIu to frozen synaptic membrane preparations in contrast with the previous findings ~2. Since the latter Ca 2÷-stimulated and C1--dependent bindings represent pharmacological characteristics similar to those found in the freshly prepared synaptic membranes, it seems possible to speculate that freezing at - 80 °C in 0.32 M sucrose may maintain the properties of membranous constituents sensitive to these ions. Although the Ca 2 ÷ -stimulated binding is not exposed in the absence of C1- ions, Ca 2 ÷ ions seem to elicit their additional facilitation of the C1--dependent binding through interacting with the anion transport carriers, rather than directly acting on the Cl--dependent binding sites of [3H]GIu in the membranes. For instance, an inhibitor of the carriers not only eliminated the C1 - -dependent binding, but also abolished the Ca 2 ÷ -stimulated binding. The Ca 2 + -stimulated binding was differentially affected by SH-reactive agents, SH-containing compounds and ascorbic acid in comparison with the CI--dependent binding. These findings along with the fact that pretreatment of the membranes with calcium acetate results in an additional potentiation of the C1--induced enhancement of the binding with a concomitant abolishment of the Ca 2 ÷ -stimulated binding 37, all support the proposal that synaptic membranes may contain 3 distinctly different binding sites such as basal, CI- -dependent and Na ÷ -dependent sites, but not Ca 2 ÷ -dependent binding sites. Calcium ions may affect some Ca 2 ÷ -sensitive membranous constituent including Ca 2 + -dependent proteases, Ca 2 + channels, cytoskeleton proteins and calmodulin systems, which in turn results in an activation of the stimulatory properties of

139 the anion transport carriers which have an interaction with the binding sites. It is conceivable that an influx of Ca 2 + ions may unmask the concealed anion carriers in the membranes since it has been demonstrated that intracellular Ca 2 + ions have the ability to open the anion channels in Xenopus oocytes23 and in the oocytes implanted with rat brain fnessenger RNA 25. The prevailing view on the functional significance of CI--dependent binding is that the binding represents an anion-driven uptake of Glu into resealed membrane vesicles, but not a linkage of Glu receptors to the anion channels 14'18,27. Although one strong piece of supporting evidence for this uptake hypothesis is the temperature dependency of the C1--dependent binding, it still seems possible that the latter temperature dependency may be derived from the activation energy of the anion channels as shown in human red cells 6. In addition, similarly DIDS-sensitive potentiation by anions has been reported to occur on the GABA/benzodiazepine/barbiturate receptors complex8,9 which undoubtedly resides at postsynaptic membranes. It has been also demonstrated that the Glu-induced hyperpolarization (H-response) is abolished by an antagonist for the anion channels in the locust neuromuscular junction ~°. The antagonist as well as inhibitors of anion transport (DIDS and ethacrynic acid) invariably diminished the CI--dependent binding in the rat synaptic membranes 36. From this point of view, it should be noted that Glu elicited oscillatory membrane currents carried by CI- ions which were triggered possibly by intracellular Ca 2 + ions in Xenopus oocytes injected with messenger RNA extracted from the rat brain 25. Whether or not C1-- and C1- / C a 2 + -induced uptake of this potential neurotransmitter across the synaptic membranes really occurs in the absence o f N a + ions in a DIDS-sensitive manner, however, should be evaluated in future studies before drawing a conclusion. It is noteworthy that the binding rapidly reaches its maximal value with a progressive decline in the presence of Na + ions. This may be attributable to the facilitated dissociation of [3H]Glu from its binding sites in the presence of 100 mM sodium acetate24. Several lines of evidence have indicated the conformational transformation of various membranous proteins by sodium ions such as opiate receptors in synaptic membranes 26 and amino acid transport proteins in erythrocyte membranes 7. These previous findings together with the present results that Na + ions affect the alTmity of the [3H]Glu binding sites with a concurrent alteration of their density, maintain the hypothesis that Na + ions may expose the binding sites of [ 3H]Glu intrinsically different from the receptor sites. Pharmacological characterization of the individual binding sites using D-Asp, DL-AP-4 and QA all support the above assumption. It should be emphasized that SH-reactive agents, SH-containing compounds and ascorbic acid invariably affected differentially the CI--dependent binding and Ca 2 +stimulated binding. Ascorbic acid has been shown to induce to a significant alteration of various neurotransmitter receptor bindings ~9 such as dopamine, serotonin, opiate and noradrenaline possibly through the peroxidation of membrane lipids. Although the exact molecular mechanisms underlying the ascorbate-induced selective elimination of the Ca 2 ÷-stimulated binding are uncertain at present, the data provided in this study

140 all raise the possibility that action sites o f C a 2 ÷ ions m a y be m e m b r a n o u s constituent which have no significant affinity for the ligand, but not the C 1 - - d e p e n d e n t sites of [ 3 H ] G l u binding. Calcium ions m a y alter the stimulatory properties of the anion t r a n s p o r t carriers which have an interaction with the [ 3 H ] G l u binding sites. This is 'also s u p p o r t e d by the differential effect o f SH-reactive agents and S H - c o n t a i n i n g c o m p o u n d s on the C 1 - - d e p e n d e n t a n d C a 2 ÷ -stimulated bindings. It thus a p p e a r s that C a 2 ÷ ions are able to potentiate the CI - - d e p e n d e n t portion of [3H ]Glu binding through interacting with the D I D S-sensitive anion t r a n s p o r t carriers. It is conceivable that the latter interaction m a y in part participate in the exhibition o f various p s y c h o t r o p i c actions by centrally acting drugs k n o w n to induce a significant m o v e m e n t o f C a 2 ÷ ions across the synaptic m e m b r a n e s .

ACKNOWLEDGEMENT This w o r k was s u p p o r t e d in p a r t by a G r a n t 61770161 to Y.Y. from the Ministry o f Education, Science a n d Culture, J a p a n .

REFERENCES 1 All, F., Nachbar, M.J. and Oppenheim, J.D., Chloride self exchange in Ehrlich cells: inhibition by furosemide and 4-acetamide-4'-isothiocyanato-stilbene-2-2'-disulfonic acid, Biochem. Biophys. Acta, 471 (1977) 341-347. 2 Baudry, M. and Lynch, G., Regulation of glutamate receptors by cations, Nature (London), 282 (1979) 748-750. 3 Baudry, M. and Lynch, G., Regulation of hippocampal glutamate receptors: Evidence for the involvement of a calcium activated protease, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 2298-2302. 4 Baudry, M., Smith, E. and Lynch, G., Influences of temperature, detergents and enzymes on glutamate receptor binding and its regulation by calcium in rat hippocampal membranes, Mol. Pharmacot., 20 (1981) 280-286. 5 Baudry, M., Bundman, M.C., Smith, E.K. and Lynch, G., Micromolar calcium stimulates proteolysis and glutamate binding in rat brain synaptic membranes, Science, 212 (1981) 937-938. 6 Brahm, J., Temperature-dependent changes of chloride transport kinetics in human red cells, J. Gen. Physiol., 70 (1977) 283-306. 7 Charalambous, B.M. and Wheeler, K.P., Sodium-induced conformation changes in membrane transport proteins, FEBS Lett., 189 (1985) 163-166. 8 Costa, T., Rodbard, D. and Pert, C.B., Is benzodiazepine receptor coupled to a chloride anion channel?, Nature (London), 277 (1979) 315-317. 9 Costa, T., Russel, L., Pert, C.B. and Rodbard, D., Halide- and ~-aminobutyric acid-induced enhancement of diazepam receptors in rat brain. Reversal by disulfonic stilbene blockers of anion channel, Mol. Pharmacol., 20 (1981) 470-476. 10 Cull-Candy, S.G. and Usherwood, P.N.R., Two populations of L-glutamate receptors on locust muscle fibers, Nature New Biol., 249 (1973) 62-64. 11 Curtis, D.R., and Johnston, G.A.R., Amino acid transmitter in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 94-188.

141 12 Fagg, G.E., Mena, E.E., Monaghan, D.T. and Cotman, C.W., Freezing eliminates a specific population of L-glutamate receptors in synaptic membranes, Neurosci. Lett., 38 (1983) 157-162. 13 Fagg, G.E., Foster, A.C., Mena, E.E. and Cotman, C.W., Chloride and calcium ions separate L-glutamate receptor populations in synaptic membranes, Eur. J. PharmacoL, 88 (1983) 105-110. 14 Fagg, G.E. and Matus, A., Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6876-6880. 15 Fagg, G.E., Riederer, B. and Matus, A., Sodium ions regulate a specific population of acidic amino acid receptors in synaptic membranes, Life Sci., 34 (1984) 1739-1745. 16 Fonnum, F., Glutamate: a neurotransmitter in mammalian brain, J. Neurochem., 42 (1984) 1-11. 17 Foster, A.C. and Roberts, P.J., High affinity L-[3H]glutamate binding to post-synaptic receptor sites on rat cerebellar synaptic membranes, or. Neurochem., 31 (1978) 1467-1477. 18 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 19 Jones, S.B. and Bylund, D.B., Ascorbic acid inhibition of alpha-adrenergic receptor binding, Biochem. Pharmacol., 35 (1986) 595-599. 20 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 21 Mena, E.E., Fagg, G.E. and Cotman, C.W., Chloride ions enhance L-glutamate binding to rat brain synaptic membranes, Brain Res, 243 (1982) 378-381. 22 Michaelis, E.K., Michaelis, M.L. and Boyarsky, L.L., High-affinity glutamic acid binding to brain synaptic membranes, Biochim. Biophys. Acta, 367 (1974) 338-348. 23 Miledi, R., A calcium-dependent transient outward current in Xenopus laevis oocytes, Proc. R. Soc. London Ser. B, 215 (1982) 491-497. 24 Ogita, K. and Yoneda, Y., Characterization ofNa + -dependent binding sites of [3H]glutamate in synaptic membranes from rat brain, Brain Res., in press. 25 Parker, I., Gunderson, C.B. and Miledi, R., Intracellular Ca2+-dependent and Ca2÷-independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes, Neurosci. Res., 2 (1985) 491-496. 26 Pert, C.B. and Snyder, S.H., Opiate receptor binding of agonists and antagonists affected differentially by sodium, Mol. Pharmacol., 10 (1974) 868-879. 27 Pin, J.-P., Bockaert, J. and Recasesn, M., The Ca2+/C1 - dependent t-[3H]glutamate binding: a new receptor or a particular transport process?, FEBS Lett., 175 (1984) 31-36. 28 Roberts, P.J., Glutamate receptors in the rat central nervous system, Nature (London), 252 (1974) 399-401. 29 Ross, A.H. and McConnel, H.M., Reconstitution of the erythrocyte anion channel, J. Biol. Chem., 253 (1978) 4777-4782. 30 Takahashi, M., Yoneda, Y. and Kuriyama, K., Enhancement of 7-aminobutyric acid (GABA) receptor binding by lipophilic antioxidants, Brain Res., 296 (1984) 164-167. 31 Vincent, S.R. and McGeer, E., A comparison of sodium-dependent glutamate binding with high-affinity glutamate uptake in the rat striatum, Brain Res., 184 (1980) 99-108. 32 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 33 Yoneda, Y. and Kuriyama, K., Presence of a low molecular weight endogenous inhibitor on 3H-muscimol binding in synaptic membranes, Nature (London), 285 (1980) 670-673. 34 Yoneda, Y. and Kuriyama, K., Some characteristics of [3H]muscimol binding to synaptic membrane from rat brain, Brain Res., 197 (1980) 554-560. 35 Yoneda, Y., Kuriyama, K. and Takahashi, M., Modulation of synaptic GABA receptor binding by membrane phospholipids: possible role of active oxygen radicals, Brain Res., 333 (1985) 111-122.

142 36 Yoneda, Y., Ogita, K., Nakamuta, H., Koida, M., Ohgaki, T. and Meguri, H., Possible interaction ,~" [3H]glutamate binding sites with anion channels in rat neural tissues, Neurochem. Int., in press. 37 Yoneda, Y. and Ogita, K., Are CaZ+-dependent proteases really responsible for CI- -dependent and Ca 2 ÷ -stimulated binding of [3H]glutamate in rat brain?, Brain Res., in press. 38 Yoneda, Y. and Ogita, K., [3H]Glutamate binding sites in the rat pituitary, Neurosci. Res.. 3 (1986) 430-435. 39 Zukin, S.R., Young, A.B. and Snyder, S.H., Gamma-aminobutyric acid binding to receptor sites in the rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., (1974)4802-4807.