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barbiturate receptor complex
Neurophormacology
Printed ~aGreat
Vol.
23,No. 7B,pp.853-854, 1984
0028.3908184 $3.00 + 0.00
Britain
PURIFICATION
R.W. Olsen,
Pergamon
OF THE GABA/BENZODIAZEPINE/BARBITURATE
J.B. Fischer,
R.G. King, J.Y. Ransom
RECEPTOR
Press Ltd
COMPLEX
and G.B. Stauber
Department of Pharmacology, School of Medicine, and Brain Research Institute, University of California, Los Angeles, and Department of Biochemistry, University of California,Riverside, CA, USA
The synaptic receptor for the inhibitory neurotransmitter post y-aminobutyric acid (GABA) is a complex protein containing a chloride channel modulatory sites for two classes of drugs, the and Barbiturates which benzodiazepines (BZ) and convulsants/barbiturates. tissue and enhance GABA-activated chloride permeability at the cellular level also are able to enhance benzodiazepine and GABA receptor binding at the molecular vel. Convulsant/barbiturate sites can be assayed directly with [ 3seS]t-butyl bicyclophosphorothionate (TBPS). This activity was solubilized with the detergent CHAPS and shown to co-migrate with BZ and GABA receptor binding activity during protein fractionation as a single protein complex, which could be purified intact by over lOOO-fold on benzodiazepine affinity columns. of chloride channels via of action of GABA involves regulation The major mechanism receptors with specificity for the antagonist bicuculline and the agonist muscimol. These chloride channel-mediated inhibitory synapses are inhibited by picrotoxin and related We have developed an barbiturates and related depressants. ~o,~~~l~rt"GA~~~a~~~~~~~~ @ Cl flux in rat brain slices (Wong et al., 1984a). This GABA barbiturates (1,3_dimethylbutyl barbiturate, function is enhanced by anesthetic pentobarbital. (+)hexobarbital) and related depressants, e.g. etazolate and (+)etomidate, but not by inactive analogs ((-)hexobarbital and (-)etomidate). These same depressant drugs allosterically enhance the affinity of BZ binding in rat brain membranes in vitro. The effect: is chemically specific and stereospecific; requires the which permeate GABA ion channels; is blocked of chloride or other anions presence involves the same depress s which at competitvely by picrotoxin and related convulsants; similar concentrations inhibit the binding of radioactive picrotoxin or [%s]TBPS; is blocked allosterically by the GABA antagonist bicuculline (Leeb-Lundberg et al. 1980; Likewise, these Leeb-Lundberg et al. Leeb-Lundberg and Olsen, same 1981; 1982). depressants in a picrotoxin-sensitive manner allosterically inhibit the binding of the BZ "inverse agonist" B-carboline-3-carboxylate methyl ester (Wang et al. 198413). enhance in a chloride-dependent Similarly, barbiturates and etazolate and these picrotoxin-sensitive manner the binding of GABA receptor agonists (Olsen and Snowman, 1982) by increasing the affinity of "super-low affinity" (KD = 1 uM) agonist sites (Olsen et al. Super-low affinity agonists sites can be conveniently labeled with high affinity 1984a). using a radioactive antagonist [sH]bicuculline methochloride (Olsen and Snowman, 1983). These sites have been found by brain slice autoradiography to be localized to the same brain regions as BZ binding sites but to have some differences from [3H]muscimol binding, especially in cerebellum (Olsen et al. 1984b). Consistent with the allosteric model of agonist-antagonist states for the GABA receptor (Olsen et al. 1984a), [3H]bicuculline methochloride binding is allosterically inhibited by the barbiturates which enhance agonist binding (Wong et al. 1984b). This evidence shows that the GABA/BZ receptor complex contains a modulatory receptor site for barbituates. GABA and BZ receptor binding activities can be solubilized with mild detergents such as the zwitterionic3gHAPS. Barbiturate enhancement is maintained and convulsant/barbiturate-displaceable [ S]TBPS binding activity is also solubilized. All of these activities co-migrate on various protein fractionation procedures as a single protein complex (King and Olsen, 1984).
853
854
R. w. OLSEN er al.
all three activities are retained on a benzodiazepine affinity column and Furthermore, [3H]M~~~i~~lbinding activity is purified over IOOO-fold. co-purified by free BZ elution. labeled with [ 3H] f lunitrazepam following The purified material can be photo-affinity and shows a single radioactive peak on gel filtration columns ion-exchange chromatography, A single radioactive peak of similar with an apparent molecular weight of abo 200,000. %5T size was seen following iodination with . SDS gel electrophoresis revealed 1-5 bands in the molecular weight region of 56-66,000, and [3H]flunitrazepam was associated with one of these. Thus the GABA receptor-chloride ion channel protein with associated modulatory sites for benzodiazepines and for convulsants/barbiturates can be solubilized and purified. REFERENCES of convulsant/barbiturate binding activity King R.G. and Olsen R.W. (1984) Solubilization on the GABAlbenzodiazepine receptor complex. Biochem. Biophys. Res. Comm. in press. ALL-Leeb-Lundberg F., Snowman A. and Olsen R.W. (1980) Barbiturate receptors are coupled to benzodiazepine receptors. Proc. Natl. Acad. Sci. USA 77: 7468-7472. ------Leeb-Lundberg F., Snowman A. and Olsen R.W. (1981) Perturbation of benzodiazepine receptor binding by pyrazolopyridines involves picrotoxinin/barbiturate receptor sites. J. Neurosci. 1: 471-477. Leeb-Lundberg-F. and Olsen R.W. (1982) Interactions of barbiturates of various pharmacological categories with benzodiazepine receptors. Mol. Pharmacol. 21: 320-328. Olsen R.W. and Snowman A.M. (1982) Chloride-dependent enhancemz by barbituracs of GABA enhanrement by barbiturates of GABA receptor binding. .J. Neurosci. 2: 1912-1823. Olsen R.W. and Snowman A.M. (1983) [3H]Bicuculline methochioride bindin; to low-affinity y-aminobutyric acid receptor sites. J. Neurochem. 41: 1653-1663. Olsen R.W., Wong E.H.F., Stauber G.B. and King R.G. (1984a) Biochemical pharmacology of the GABA/benzodiazepine receptorlionophore protein, Fed. Proc. in press. -Olsen R.W., Snowhill E.W. and Wamsley J.K. (1984b) Autoradiographic localization of low affinity GABA receptors with [3H]bicuculline methochloride. Eur. J. Pharmacol. -in -press. Wang E.H.F., Leeb-Lund~~rg_~.~.F., Teichberg V.I. and Olsen R.W. (1984a) y-Aminobutyric acid activation of Cl. flux in rat hippocampal slices and its potentiation by barbiturates. Brain Res. in press. --k'ong E.H.F., Snowman A.M., Leeb-Lundberg L.M.F. and Olsen R.W. (1984b) Barbiturates allosterically inhibit GABA antagonist and benzodiazepine inverse agonist binding. -Eur. in press. -J. Pharmacol. --
Printed ~aGreat
Vol.
23,No. 7B,pp.853-854, 1984
0028.3908184 $3.00 + 0.00
Britain
PURIFICATION
R.W. Olsen,
Pergamon
OF THE GABA/BENZODIAZEPINE/BARBITURATE
J.B. Fischer,
R.G. King, J.Y. Ransom
RECEPTOR
Press Ltd
COMPLEX
and G.B. Stauber
Department of Pharmacology, School of Medicine, and Brain Research Institute, University of California, Los Angeles, and Department of Biochemistry, University of California,Riverside, CA, USA
The synaptic receptor for the inhibitory neurotransmitter post y-aminobutyric acid (GABA) is a complex protein containing a chloride channel modulatory sites for two classes of drugs, the and Barbiturates which benzodiazepines (BZ) and convulsants/barbiturates. tissue and enhance GABA-activated chloride permeability at the cellular level also are able to enhance benzodiazepine and GABA receptor binding at the molecular vel. Convulsant/barbiturate sites can be assayed directly with [ 3seS]t-butyl bicyclophosphorothionate (TBPS). This activity was solubilized with the detergent CHAPS and shown to co-migrate with BZ and GABA receptor binding activity during protein fractionation as a single protein complex, which could be purified intact by over lOOO-fold on benzodiazepine affinity columns. of chloride channels via of action of GABA involves regulation The major mechanism receptors with specificity for the antagonist bicuculline and the agonist muscimol. These chloride channel-mediated inhibitory synapses are inhibited by picrotoxin and related We have developed an barbiturates and related depressants. ~o,~~~l~rt"GA~~~a~~~~~~~~ @ Cl flux in rat brain slices (Wong et al., 1984a). This GABA barbiturates (1,3_dimethylbutyl barbiturate, function is enhanced by anesthetic pentobarbital. (+)hexobarbital) and related depressants, e.g. etazolate and (+)etomidate, but not by inactive analogs ((-)hexobarbital and (-)etomidate). These same depressant drugs allosterically enhance the affinity of BZ binding in rat brain membranes in vitro. The effect: is chemically specific and stereospecific; requires the which permeate GABA ion channels; is blocked of chloride or other anions presence involves the same depress s which at competitvely by picrotoxin and related convulsants; similar concentrations inhibit the binding of radioactive picrotoxin or [%s]TBPS; is blocked allosterically by the GABA antagonist bicuculline (Leeb-Lundberg et al. 1980; Likewise, these Leeb-Lundberg et al. Leeb-Lundberg and Olsen, same 1981; 1982). depressants in a picrotoxin-sensitive manner allosterically inhibit the binding of the BZ "inverse agonist" B-carboline-3-carboxylate methyl ester (Wang et al. 198413). enhance in a chloride-dependent Similarly, barbiturates and etazolate and these picrotoxin-sensitive manner the binding of GABA receptor agonists (Olsen and Snowman, 1982) by increasing the affinity of "super-low affinity" (KD = 1 uM) agonist sites (Olsen et al. Super-low affinity agonists sites can be conveniently labeled with high affinity 1984a). using a radioactive antagonist [sH]bicuculline methochloride (Olsen and Snowman, 1983). These sites have been found by brain slice autoradiography to be localized to the same brain regions as BZ binding sites but to have some differences from [3H]muscimol binding, especially in cerebellum (Olsen et al. 1984b). Consistent with the allosteric model of agonist-antagonist states for the GABA receptor (Olsen et al. 1984a), [3H]bicuculline methochloride binding is allosterically inhibited by the barbiturates which enhance agonist binding (Wong et al. 1984b). This evidence shows that the GABA/BZ receptor complex contains a modulatory receptor site for barbituates. GABA and BZ receptor binding activities can be solubilized with mild detergents such as the zwitterionic3gHAPS. Barbiturate enhancement is maintained and convulsant/barbiturate-displaceable [ S]TBPS binding activity is also solubilized. All of these activities co-migrate on various protein fractionation procedures as a single protein complex (King and Olsen, 1984).
853
854
R. w. OLSEN er al.
all three activities are retained on a benzodiazepine affinity column and Furthermore, [3H]M~~~i~~lbinding activity is purified over IOOO-fold. co-purified by free BZ elution. labeled with [ 3H] f lunitrazepam following The purified material can be photo-affinity and shows a single radioactive peak on gel filtration columns ion-exchange chromatography, A single radioactive peak of similar with an apparent molecular weight of abo 200,000. %5T size was seen following iodination with . SDS gel electrophoresis revealed 1-5 bands in the molecular weight region of 56-66,000, and [3H]flunitrazepam was associated with one of these. Thus the GABA receptor-chloride ion channel protein with associated modulatory sites for benzodiazepines and for convulsants/barbiturates can be solubilized and purified. REFERENCES of convulsant/barbiturate binding activity King R.G. and Olsen R.W. (1984) Solubilization on the GABAlbenzodiazepine receptor complex. Biochem. Biophys. Res. Comm. in press. ALL-Leeb-Lundberg F., Snowman A. and Olsen R.W. (1980) Barbiturate receptors are coupled to benzodiazepine receptors. Proc. Natl. Acad. Sci. USA 77: 7468-7472. ------Leeb-Lundberg F., Snowman A. and Olsen R.W. (1981) Perturbation of benzodiazepine receptor binding by pyrazolopyridines involves picrotoxinin/barbiturate receptor sites. J. Neurosci. 1: 471-477. Leeb-Lundberg-F. and Olsen R.W. (1982) Interactions of barbiturates of various pharmacological categories with benzodiazepine receptors. Mol. Pharmacol. 21: 320-328. Olsen R.W. and Snowman A.M. (1982) Chloride-dependent enhancemz by barbituracs of GABA enhanrement by barbiturates of GABA receptor binding. .J. Neurosci. 2: 1912-1823. Olsen R.W. and Snowman A.M. (1983) [3H]Bicuculline methochioride bindin; to low-affinity y-aminobutyric acid receptor sites. J. Neurochem. 41: 1653-1663. Olsen R.W., Wong E.H.F., Stauber G.B. and King R.G. (1984a) Biochemical pharmacology of the GABA/benzodiazepine receptorlionophore protein, Fed. Proc. in press. -Olsen R.W., Snowhill E.W. and Wamsley J.K. (1984b) Autoradiographic localization of low affinity GABA receptors with [3H]bicuculline methochloride. Eur. J. Pharmacol. -in -press. Wang E.H.F., Leeb-Lund~~rg_~.~.F., Teichberg V.I. and Olsen R.W. (1984a) y-Aminobutyric acid activation of Cl. flux in rat hippocampal slices and its potentiation by barbiturates. Brain Res. in press. --k'ong E.H.F., Snowman A.M., Leeb-Lundberg L.M.F. and Olsen R.W. (1984b) Barbiturates allosterically inhibit GABA antagonist and benzodiazepine inverse agonist binding. -Eur. in press. -J. Pharmacol. --