Solubilization of convulsant/barbiturate binding activity on the γ-aminobutyric acid/benzodiazepine receptor complex

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.

Section: Discussionmentioning

confidence: 99%

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

Eichinger¹,

Sieghart²

1985

“…[3H]Diazepam, [3H]PCCM, and [3'S]TBPS binding was assayed by filtration on Whatman GF/B glass fiber filters, followed by washing twice with 2.5 ml of 0.2 M NaCI. The assay buffer for all three ligands was 10 mM sodium phosphate, pH 7.0, plus 0.2 M NaCI; the assay volume was 0.5 ml, containing about 0.5 mg protein (Lowry et al, 1951); and the equilibration times were 60 min at 0°C for the two BZ ligands and 90 min at 22°C for [%ITBPS (Leeb-Lundberg et al, 1981b;Wong et al, 1984;King and Olsen, 1984). Backgrounds were determined in the presence of 10 pM diazepam for BZ receptor sites and 10 p M picrotoxin for [35S]TBPS binding.…”

Section: Methodsmentioning

confidence: 99%

“…The pyrazolopyridine and barbiturate enhancement of BZ binding were reported to be chloridedependent and inhibited by picrotoxin and related GABA chloride channel antagonists, as well as by bicuculline and related GABA receptor antagonists (Supavilai et al, 1981;Asano and Ogasawara, 1981;Leeb-Lundberg et al, 1980Skolnick et al, 1982;Ticku and Maksay, 1983). Further, these depressant compounds at similar concentrations inhibited the binding of [3H]a-dihydropicrotoxinin (Olsen, 1981) and [3sS]TBPS (Squires et al, 1983;Ticku and Maksay, 1983;King and Olsen, 1984;Supavilai and Karobath, 1984) to the convulsant receptor sites. The same compounds (Olsen and Snowman, 1982;Asano and Ogasawara, 1981;Whittle and Turner, 1982;Willow and Johnston, 1983) showed a picrotoxin-sensitive, chloride-dependent enhancement of GABA receptor binding.…”

Section: Avm Is a Glycosidic Macrocyclic Lactone Frommentioning

confidence: 99%

“…Reciprocal allosteric interactions in mem-brane binding in vitro between GABA receptor ligands, BZs, and barbiturates have suggested a multiple-receptor, chloride channel model for the GABA receptor complex (Olsen, 1981). Picrotoxin and related convulsant drugs inhibit the function of this GABA receptor-chloride channel complex, and the barbiturate-sensitive convulsant receptor sites on the complex can be assayed directly with the radioactive cage convulsant [35S]t-butyl bicyclophosphorothionate (TBPS; Squires et al, 1983;Ticku and Maksay, 1983;Supavilai and Karobath, 1984;King and Olsen, 1984;Trifiletti et al, 1984).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Avermectin B_la Modulation of γ‐Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

Olsen

Snowman

1985

Self Cite

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.

“…The binding sites for the channel-gating ligands, as measured by the binding of [35S]TBPS or by the barbiturate facilitation of benzodiazepine or GABA, binding, were not detectable in the receptor extracted or purified under the aforementioned conditions (Sigel et al, 1983). It was shown, however, that when the brain membranes were extracted with the zwitterionic detergent 3-[(3-cholamidopropyI)dimethylammonio]-1 -propanesulphonate (CHAPS), both [35S]TBPS binding (King and Olsen, 1984;Trifiletti et al, 1984;Stephenson et al, 1 9 8 6~) and the barbiturate enhancement effects are found in solution . Furthermore, subsequent purification of the receptor in the presence of CHAPS and added phospholipid showed that these properties were correspondingly retained in the isolated protein from bovine (Sigel and Barnard, 1984) or porcine (Kirkness and Turner, 1986) brain.…”

mentioning

confidence: 99%

Molecular Size of the γ‐Aminobutyric Acid_A Receptor Purified from Mammalian Cerebral Cortex

Barnard

Stephenson

1989

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.