Biochemical characterization of an isolated and functionally reconstituted gamma-aminobutyric acid/benzodiazepine receptor.
We have solubilized, affinity-purified, and functionally reconstituted the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8-9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52-56, and 59-62 kDa, the latter two of which appears as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA-stimulated 36Cl- flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.
Bristow
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DR
MRC Molecular Neurobiology Unit, University of Cambridge Medical School, England.
Martin
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D000818:Animals; D001921:Brain / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D002793:Cholic Acids; D003902:Detergents; D007425:Intracellular Membranes / Q000378:metabolism; D008055:Lipids; D008081:Liposomes / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013574:Synaptosomes / Q000378:metabolism
D002712:Chlorides; D002793:Cholic Acids; D003902:Detergents; D008055:Lipids; D008081:Liposomes; D011963:Receptors, GABA-A; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate