Characterization of solubilized benzodiazepine and muscimol binding sites from rat brain

Nrurophurmu~~~lo!/?, Vol. 71. pp. 1355 to 135x. 1982 Printed in Great Brltain

00?8-390x:82/1~1355-04803.00/0 Pergamnn Press Ltd

CHARACTERIZATION OF SOLUBILIZED BENZODIAZEPINE AND MUSCIMOL BINDING SITES FROM RAT BRAIN A. YA. KORNEYEV Institute of Psychiatry

U.S.S.R. Academy of Medical Sciences. I 13 152. Moscow, U.S.S.R. (Accepted

I1 May

Zagorodnoye

Shosse. 2,

1982)

Summary--Binding sites for muscimol and flunitrazepam were solubilized and their properties studied by ultrafiltration. isoelectro-focusing and cation-exchange chromatography methods. The heterogenity of binding sites for flunitrazepam was demonstrated in the ultrafiltration experiments: the binding sites with a molecular weight of more than 300,000 dalton were sensitive to stimulation by y-aminobutvric acid (GABA) and those with a molecular weight of less than 300,000 dalton were not. The binding sites for flunitrazepam appeared to be low acidic molecules with pI 5.G6.0 and binding sites for muscimol an apparently more heterogenous distribution in the range of pH 4.6 to 5.9 was detected. Cation-exchange chromatography revealed two subpopulations of binding sites for muscimol one of which copurified together with binding sites for flunitrazepam. Each subpopulation exhibited only one class of binding site with Kd = 20 nM for a subpopulation copurifying together with binding sites for flunitrazepam and K,, = 9 nM for the other.

A considerable amount of pharmacological data indicates tight interaction between benzodiazepine and y-aminobutyric acid (GABA)-receptors in the brain (Iversen, 1978; Tallman. 1980). This evidence poses the question as to whether receptors for benzodiazepines and GABA are structurally bound in a single supramolecular complex in the cell membrane. Recently this hypothesis was supported biochemically by copurification of receptors for benzodiazepines and GABA solubilized from brain membranes on the benzodiazepine affinity column (Gavish and Snyder, 1981) and by the demonstration of the enhancement by GABA of the affinity of benzodiazepine receptors in the solubilized preparation (Olsen. 1981; Gavish and Snyder, 1981). However, it is also possible to obtain the solubilized preparation from brain membranes where the benzodiazepine receptors are independent of GABA and not bound to GABA receptors (Yousufi. Thomas and Tallman, 1979). In the present work an attempt is made to demonstrate the presence of a GABA-benzodiazepine receptor complex as GABA and benzodiazepine receptors. independently, in the same solubilizate from the rat brain.

METHODS

The membranes from rat brain were prepared by triple washing of the brain homogenate (homogenization, centrifugation-30,000 8, 4”C, 30 min and rehomogenization) in 50 vol. of ice-cold 50 mM Tris-HCl Key binding

words: benzodiazepine binding sites, receptors solubilization.

sites,

muscimol

buffer, pH 7.3 at 20°C (Buffer TFmembrane A type. In some experiments triple-washed membranes were additionally frozen-thawed three times and washed in 50 vol. of ice-cold buffer T after each thawingmembranes B type. After the last centrifugation the pellets were resuspended in 10~01. of buffer T and IO’+, solution of Triton X-100 (Merck) was added to obtain a final concentration of 2’/,. The mixture was incubated on the magnetic stirrer for an hour at room temperature (25’C) and centrifuged at 120,000~ and 10°C for 90 min. The supernatant was collected and filtered through a 0.22 mkM pore size filter (GS type, Millipore) with prefilter AP-15 (Millipore) and filtrates referred to as solubilized sample A (prepared from only washed membranes type A) or solubilized sample B (prepared from washed, triple frozen-thawed membranes type B) were concentrated on the ultrafilter UM-2OE (Amicon) 5-IO-fold. The properties of the binding sites for benzodiazepine and muscimol present in these preparations were studied by the ultrafiltration methods, preparative isoelectrofocusing and cation-exchange chromatography. The specific binding C3H]flunitrazepam and C3H] muscimol was determined as described by Yousufi et al.. (1979). In brief, the samples were incubated with the radioactive ligand in 0.5 ml of buffer T with 0.5% Triton X-100 at 4°C for 1 hr. Then 0.3 ml of 2% bovine gamma-globulin and 0.3 ml of 40% polyethylene glycol 6000 (w/v) were added. The assay mixtures were rapidly vortexed and, following the addition of 3 ml of 7% polyethylene glycol, immediately filtered through GF/B filters under suction. The filters were washed rapidly with lOm1 of ice-cold 7% polyethyl-

1355

1356

A. YA. KORNEYEV

ene glycol and the trapped radioactivity was counted in 8 ml of dioxan-based scintillation mixture. Specific binding was calculated as the difference between the total binding of [3H]funitrazepam or [3H]muscimol and the binding in the presence of 0.01 mM diazepam or 1 mM of GABA respectively.

(a)

RESULTS

Incubation of the membrane suspension in 2”, Triton X-100 solution provided the solubilization of 6&70”. of the benzodiazepine binding sites in both types of membrane preparations. Under this condition. 50”,, of the muscimol binding sites was solubilized from triple frozen-thawed membranes (solubilizate B). It was difficult to estimate the degree of the solubilization of the muscimol binding sites in the case of the solubilized sample A due to the presence of potent inhibitors in the tissue homogenate. 4

The muscimol binding sites present in both types of solubilized samples were fully trapped by the ultrafilters XM-100 (cut off level 100,000 dalton) and XM-300 (cut off level 300,000 dalton). At the same time 40-45”,, of benzodiazepine binding sites present in the solubilized sample A and l&-15”, of benzodiazepine binding sites in solubilized sample B penetrated through the ultrafilter XM-300. Ten per cent of the benzodiazepine binding sites in solubilized sample A also penetrated through the XM-100 ultrafilter. It is important to note that a new ultrafilter was used in each experiment as the permeability of these ultrafilters for benzodiazepine binding sites rapidly decreased to zero during the filtration.

5

6

7

6

PH

Fig. 2. Preparative flat-bed electrofocussing in the granulated gel Ultrodex (LKB) of the soluhilizates A (A) and B (B). The solubilizate under investigation was concentrated lo-fold on the ultrafilter XM-50 and dialyzed overnight against IO mM ,TrissHCI buffer (pH 7.0) containing 0.2”,, Triton X-100. Ten ml of the concentrated solubilizate was applied with the help of a sample applicator (LKB) in the mihdle of the gel bed (24 cm iong). prepared from 5 g of Ultrodex and containing Triton X-100. 0.5”,, and the mixture of Amfolines in the following proport&t: pH range 446.5. 5 ml, pH range 558. 2 ml. pH range 3-10. I ml. The separation was finished after I6 hr at 8 W constant power and 5 C. The pH gradient was measured with the aid of a surface glass electrode. The 30 zones along the gel were collected and eluted with IO ml of buffer T containing O.S”,, Triton X-100. The pH in each eluate was adjusted to 7.3 with 50 mM TrisOH or IO mM HCI. ( -t[‘H]Runitrarepam (5 nM) specific binding. (---) -[“H]muscimol (50 nM) specific binding,

As seen from Figure I. GABA stimulated [“HIflunitrazepam binding to the XM-300 non-permeating benzodiazepine binding sites but not to the XM-300 permeating benzodiazepine binding sites: similar results were obtained when using muscimol.

-7 GABA

Fig. I. Effect of

-6

concentration

-4

-5 ,

tog

M

GABA on the specific binding of [‘HIflunitrazepam to the XM-300 ultrafilter permeating (0) and nonpermeating ( x ) binding sites. Each point is the mean of three experiments. The SEM was less than lo”,, of the mean value. Binding was determined in the presence of 0.5 nM [3H]flunitrazepam (FNZ).

Figure ?a and b, show the profile of [“H]muscimol and [‘H]flunitrazepam binding after the preparative isoelectrofocussing of solubilized samples A and B respectively. The main peak of bensodiazepine binding sites was revealed in the fractions with pH 5.776.0 (solubilized sample A) and 5.45.X (solubilized sample B). The distribution of the muscimol binding sites after isoelectrofocusing was more heterogenous; the highest concentration in the case of solubilized sample A was observed in the fractions with pH 5.4-5.9 with the shoulder of binding on the lower pH side (pH 4.7-5.4); in the case of solubilized sample

Solubilized 3-

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1357

and benzodiazepine receptors

GABA

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30

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50

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Fig. 3. CM-Sepharoze 4B column chromatography of solubilizate A. Fifty ml of solubilizate A was concentrated IO-fold on the XM-50 ultrafilter (Amicon) and diluted 3-fold with 50 mM succinate buffer adjusted to pH 5.5 with TrisOH and containing 0.2”” Triton X-100. The pH of this solution was adjusted to 5.5 with 50 mM succinic acid. The Solution was applied to a column (1 x 20 cm) of CMSepharose 4B. The column was washed with 100 ml of the succinate buffer (pH 5.5) containing 0.2”” Triton X-100 follow,ed by 500ml of a linear gradient of KC1 from 0 to 0.3 M (15 ml/hr) in the same buffer. Ten ml fractions were collected and the pH in each fraction adjusted to 7.3-7.5 with 50 mM Tris OH. (---tSpecitic C3H]flunitrazepam binding. (-&Specific [‘Hlmuscimol (50 nM) binding. I...)-The concentration of KCl.

B. the binding was detected pH 4.6-5.9 (Fig. 2a and b).

in the fractions

with

To try to separate the benzodiazepine receptor bound and unbound muscimol binding sites, cation exchange chromatography of the solubilized samples obtained was performed on the CM-Sepharose (Pharmacia) at the pH 5.5. As seen from Figure 3. in the case of solubilized sample A, the bulk of the [3H]muscimol binding sites was eluted in the nonadsorbed fractions, where only traces of the flunitrazepam binding sites were detected. The bulk of [3H]flunitrazepam binding was eluted at the potassium chloride concentrations above 0.10 M where muscimol binding sites were also present-fractions 30-45 (Fig. 3). These fractions were pooled and concentrated 5-fold on the ultrafilter UM 20E (Amicon) and dialyzed against buffer T with OSO,;,Triton X-100 overnight. The same procedure was applied to nonadsorbent fractions. The [3H]muscimoi specific binding with both types of pooled fractions was proportional to protein concentration (not shown) indicating the absence of an endogenous ligand. The Scatchard analysis of [3H]muscimol binding revealed only one class of binding sites in each type of pooled fractions with Kd = 9 nM for muscimol binding sites eluted in the nonadsorbent fractions and Kd = 20 nM for muscimol binding sites copurifying with benzodiazepine receptors (Fig. 4). The Kd for [3H]flunitrazepam specific binding in the pooled fractions 30-45 was equal to 4.5 nM and was not significantly different from the value obtained in the initial solubilizated samples A and B = 3.7 + 0.7 nM. However, it was

Specificaly

1000

1500

bound [“HI muscimol,

cpm per sample

Fig. 4. Scatchard analysis of specific binding of [‘HImuscimol in the fraction obtained after cation-exchange chromatography of solubilizate A (Fig. 3).,Specific binding was determined at different concentrations of [JH]muscimol in the range of 55250nM as described in Methods. O-Fractions 3310 (K, = 9 nM). &Fractions 3G-45 (K, = 20 nM).

impossible to demonstrate any stimulation of C3H]flunitrazepam binding in the presence of GABA (up to 2 mM) or muscimol (0.1 mM). Cation-exchange chromatography of solubihzate B (Fig. 5) also revealed two populations of muscimol binding sites, one eluted at a low salt concentration. where benzodiazepine binding was not detected and the other, copurifying together with [‘Hlflunitrazepam binding sites.

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Fig. 5. CM-Sepharose 4B column chromatography of solubilizate B. Fifty ml of solubilizate B was concentrated IO-fold on the XM-50 ultrafilter (Amicon) and diluted 3-fold with 50 mM succinate buffer adjusted to pH 5.5 with boric acid and containing O.Z>,,, Triton X-100. The pH of this solution was adjusted to 5.5 with 50 mM succinic acid. The solution was applied to a column (1 x 20 cm) of CMSepharose 4B. The column was washed with 100 ml of the succinic buffer (pH 5.5) containing 0.2% Triton X-100 followed by SOOml of a linear gradient of NaCl from 0 to 0.6 M (10 ml/hr) in the same buffer. The 10 ml fractions were collected and the pH in each fraction adjusted to 7.3-7.5 with 50 mM TrisOH. (---t-Specific [‘Hlflunitrazepam (1 nM) binding. (--)-Specific [3H]muscimol binding (50 nM). (. .tThe concentration of NaCl.

A. YA. KORNEYI-v

1358 DISCUSSION

Binding sites for benzodiazepines existing as a part of a GABA-receptor-ionophor complex with a molecular weight of 670,000 dalton (Asano and Ogasawara. 1981) and sensitive to GABA-stimulation (Gavish and Snyder, 1981; Asano and Ogasawara. 1981: Massotti, Guidotti and Costa, 1981) and binding sites for benzodiazepines with a molecular weight of 200,000~230.000 dalton which were not sensitive to GABA stimulation (Yousufi et al.. 1979: Lang. Barnard, Chang and Dolly. 1979) recently described. The presence of both types of binding sites for benzodiazepines in the same solubilizate was shown in the present study by ultrafiltration methods. One of these types with a molecular weight of more than 300,000 dalton was GABA-sensitive and other. with a molecular weight of less than 300.000 dalton, was GABAnonsensitive (Fig. 1). It is possible that these types of benzodiazepine binding sites are interconvertible i.e. the same binding site for the benzodiazepine molecule can transit from a free independent state to the complex state with the GABA-receptor and vice wrsa., In this case. the properties of benzodiazepine binding sites will depend not only on the methods of solubilization but also on the initial state of the binding sites in the membranes used. A comparison of the properties of the solubilized samples A and B support this conclusion. In some experiments the presence of binding sites for benzodiazepines which penetrate through an XM-100 ultrafilter was detected in the solubilized sample A. This observation is in agreement with the finding of the binding sites for benzodiazepine with ;I molecular weight of 100.000~1 10.000 dalton (Braestrup and Nielsen. 1980). In contrast to the benzodiazepine sites. the binding sites for muscimol do not permeate through any of the ultrafilters used in the present experiments. This observation is in agreement with the determination of the molecular weight of the GABA-receptor complex as 900,000 dalton (Greenlee and Olsen, 1979) and 670,000 dalton (Asano and Ogasawara, 1981) obtained by gel-chromatography methods. However, the determination of the molucular weight of the GABA-receptor complex by the sucrose density gradient centrifugation led to the value of 240.000 dalton (Olsen, 1981 ; Asano and Ogasawaru, 1981). Such disagreement between the values determined by the ultrafiltration or gel-chromatography methods and those determined by sucrose density centrifugation may indicate the non-globular structure of the GABA-receptor complex. lsoelectrofocusing studies revealed the heterogenity of the binding sites for muscimol (Fig. 2) which was more clearly demonstrated in the experiments with cation-exchange chromatography. One of two observed populations of binding sites for muscimol copurified together with the binding sites for benzodiazepine (Figs 3. 5). The heterogenity of the binding sites for muscimol described here is not likely to be an

artefact due to the method of tissue preparation, solubilization and subsequent fractionation as: (I) it was observed in both types of solubilized samples used: (2) different methods of column elution were used (see Figs 3 and 5); (3) Asano and Ogasawara (1981). who used different methods of tissue solubilization (Sodium dioxycholate and potassium chloride) and fractionation (anion-exchange chromatography) also detected the presence of two subpopulations of binding sites for muscimol. one copurifying with binding sites for benzodiazepine. Are these subpopulations physically different or does one type of binding site for muscimol exist in two conformations--benzodiazepine-receptor coupled and free’! The experiments of Massotti ct ctl. (I981 ) where, under different conditions. GABA-receptors were solubilized in :I complex with benzodiazepine receptors. or free from them, and the properties of the benzodiazepine receptors discussed in the present paper may be an argument in favour of the second case. In conclusion the data presented here indicate the presence of free binding sites for muscimol (GABAreceptors), binding sites for benzodiazepines and a GABA-benzodiazepine solubilized

fraction.

receptor These

complex

findings

raise

in the same the

possi-

and benzodiazepine receptors from an independent state to a supramolecular complex. Whether this transition is an important stage in the receptor function or is a soluhilization artefact. further investigation will show. bility

of the transition

of GABA

.l~~~1~~~clrtl~/r~~t~~~~~t.s~Thanks go to Drs R. R. Lideman and A. B. Polctayev for helpful discussion and assistance in the preparation of the manuscript. REFERENCES Asano, T. and Ogasawara,

N. (1981). Soluble gamma-aminohutyric acid and henzodiazepine receptors from rat cerebral cortex. L(f‘e Sci. 29: 1933200. Braestrup. C. and Nielsen. M. (1980). Multiple benzodiazepine receptors. Trrrds Neurosci. 3: 301-303. Gavish. M. and Snyder. S. H. (1981). y-aminobutyric acid and benzodiazepine receptors: Copurification and characterization. Proc. m1tr7. .4cud. Sci., U.S..4. 78: 193991941. Greenlee, D. V. and Olsen. R. W. (1979). Solubilization of GAMMA-aminobutyric acid receptor protein from mammalian brain, Biocl~ent. hiopltrs. Rus. Cowtmu~~.88: 38&387. Iversen. L. L. (1978). GABA and benzodiazepine receptors. Natwe 275: 477. Lang, B.. Barnard. E. A.. Chang. L. R. and Dolly. J. 0. (1979). Putative benzodiazepine receptor: A protein solubilized from brain. FEB.8 I&t. 104: 149-153. Massotti, M., Guidotti. A. and Costa. E. (1981). Characterization of benzodiazepine and y-aminobutyric recognition sites and their endogenous modulators. J. Ncurosci. I: 4099418. Olsen. R. W. (1981). GABA-benzodtazepine-barbiturate receptor interactions. J. .\‘~urochrru. 37, i 13. Tallman. J. F. (1980). Interaction between GABA and bcnzodiazepines: /3rck Rrs. Bull. 5: Suppl. 2. X29-832. Yousufi. M. A. K.. Thomas. J. W. andTallman. J. F. (1979). Solubilization of benzodiazepine binding site from rat cortex. L!f“ Sci. 25: 463470.