A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

Sigel, Erwin; Stephenson, F. Anne; C, Mamalaki; Barnard, Eric A.

doi:10.1016/s0021-9258(18)32319-6

Cited by 331 publications

(30 citation statements)

References 31 publications

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“…Evidence was accruing to suggest that the anxiolytic benzodiazepine class of drugs, such as diazepam, mediated their therapeutic effects via interaction with GABA A receptors, but it was unclear if this was a direct effect or if it occurred via interaction with an intermediary protein. Erwin Sigel in Eric's group exploited the fortuitous possible existence of a benzodiazepine binding site within the GABA A receptor to isolate to homogeneity, by benzodiazepine affinity chromatography, a multi-subunit protein consisting of α and β subunits that was shown to have all the pharmacological properties of a GABA A receptor (26, 28). Importantly, upon reconstitution in lipid bilayers, this isolated protein was found to contain an integral GABA-gated anion channel (31).…”

Section: The Ligand-gated Ion Channel Superfamilymentioning

confidence: 99%

Eric A. Barnard. 2 July 1927—23 May 2018

Stephenson

2020

Biogr. Mems Fell. R. Soc.

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Eric Barnard was a protein biochemist who played a leading role in the delineation of the molecular components of neuromuscular transmission and the emergence of molecular neuroscience as a scientific discipline. He began his career at King's College London, moving to the State University of Buffalo, New York, in 1965 before returning to Imperial College, London, in 1975. In 1985 he became the Director of the Medical Research Council (MRC) Molecular Neurobiology Unit in Cambridge. Upon retirement from the MRC, he moved to the Royal Free Hospital in London where he continued as Director of Molecular Neurobiology, but in 1998 returned to the University of Cambridge (Department of Pharmacology) as Emeritus Professor. In 2014, at the age of 86, he finally retired from active research. Although Eric was elected FRS for his early pioneering work on the protein chemistry of enzymes and the nicotinic acetylcholine receptor, his seminal contribution, initiated during his time at Imperial, was the application of molecular biological methods to the study of many neurotransmitter receptors. With Ricardo Miledi FRS (and later David Brown FRS and colleagues), he developed the Xenopus oocyte system for the expression of receptors from total tissue mRNA. His was the first group to clone a neurotransmitter receptor subunit cDNA, the nicotinic acetylcholine receptor α subunit of Torpedo marmorata . This was followed by purification and subsequent cloning of inhibitory γ-aminobutyric acid (GABA) A receptor subunit cDNAs. This achievement, driven by Eric and aided by his collaborator Peter Seeburg, led to the discovery of the ligand-gated ion channel superfamily, the discovery of neurotransmitter receptor heterogeneity, and the development of concepts of receptor families and superfamilies. His pioneering work was pivotal for the foundation of modern central nervous system drug discovery.

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Section: The Ligand-gated Ion Channel Superfamilymentioning

confidence: 99%

Eric A. Barnard. 2 July 1927—23 May 2018

Stephenson

2020

Biogr. Mems Fell. R. Soc.

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“…In agreement with the membrane-bound and crude soluble receptor preparations, the pharmacological specificities of both the purified GABA and BZ binding sites were consistent with the GABA. and the central BZ receptor specificities (Sigel et al_., 1983). GABA regulation of BZ binding activity was also demonstrated in.…”

Section: CLmentioning

confidence: 93%

“…The affinity resin was washed extensively and eluted biospecifically with either chlorazepate (Martini et al, 1982;Sigel et al_., 1982;Schoch and Mohler, 1983), flurazepam (Olsen et al_., 1984;Tallman, 1984) or the water insoluble BZ 1012S (Taguchi and Kuriyama, 1984). The eluting ligand was then removed by ion exchange chromatography (Sigel et al_., 1983;Olsen et al_., 1984), gel filtration chromatography (Schoch et al_., 1984) or dialysis (Martini et al_., 1982).…”

Section: Receptor Purificationmentioning

confidence: 99%

“…GABA regulation of BZ binding activity was also demonstrated in. the purified preparations (Martini et al_., 1983;Sigel et al_., 1983;Sigel and Barnard, 1984;Kirkness and Turner, 1986).…”

Section: CLmentioning

confidence: 99%

“…The purified receptor was found to be an acidic protein (pi = 5.5, Barnard et al_., 1984) and to have a mul ti-subunit structure. Sodium dodecyl sul phate-polyacryl amide gel electro phoresis (SDS-PAGE) under reducing conditions showed in general that the receptor purified from bovine cortex and cerebellum, rat and pig consisted of two major polypeptides with M 49,000-53,000 and M 55,000-58,000 respectively (Sigel et al_., 1983;Sigel and Barnard, 1984;Taguchi and Kuriyama, 1984;Kirkness and Turner, 1986). However, the group of Martini reported a single subunit with M 60,000 (Martini et al_., 1983) and the group of Olsen four subunits with M f values of 56,000, 60,000, 61,000, and 66,000 for purified rat receptor (Olsen e_t al_., 1984).…”

Section: Purified Receptor Characterisationmentioning

confidence: 99%

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Studies on the Brain Γ-Aminobutyric Acid/Benzodiazepine Receptor and Its Antibodies

Μαμαλακη¹

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Chapter 1 Introduction 1.1 6ABA receptor measurement in vitro 1.2 Benzodiazepine receptor measurement in vitro 1.3 GABA-BZ receptor interactions 11 1.4 Convulsant, depressant and Picrotoxin binding sites and their interactions with the GABA/BZ receptor 1.5 Molecular characterisation of the GABA/BZ receptor 16 1.5.1 Membrane studies 1.5.2 Solubil isation of the GABA/BZ complex 1.5.3 Receptor purification 21 1.5.4 Purified receptor characterisation 25 1.5.5 GABA/BZ receptor antibodies 1.6 The purpose of the present study 29 Chapter 2 Studies on the soluble, purified and reconstituted GABA/BZ receptor complex 31 2.1 Materials 2.2 Methods 2.2.1 Synthesis of the affinity chromatography col umn Page 2.3.2 Hydrodynamic properties of the soluble and purified GABA/BZ receptor complex 81 2.3.2.1 Sucrose density gradient sedimentation of the Ν a-de oxy chol ate solubilised GABA/8Z receptor 81 2.3.2.2 Gel filtration of the Na-deoxycholate solubilised receptor complex 92 2.3.2.3 Molecular weight determination of the Na-deoxycholate solubilised receptor compi ex 98 2.3.2.4 Hydrodynamic properties of the CHAPS solubilised GABA/BZ receptor complex 106 2.3.2.5 Gel filtration chromatography of the Na-deoxychoi ate/Triton X-100 purified GABA/BZ receptor 119 2.3.2.6 Sucrose density gradients of the Na-deoxychoi ate/Triton X-100 purified GABA/BZ receptor complex 120 2.3.3 Reconstitution of the GABA/BZ receptor into phosphol ipid vesicles 137 2.4 Discussion 150 2.4.1 Receptor solubilisation and purification 150 2.4.2 Hydrodynamic properties of the soluble and purified GABA/BZ receptor complex 154 2.4.3 Reconstitution of the purified receptor into 1 ipid vesicles 161

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Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

et al. 1998

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The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of gamma-aminobutyric acid (GABA) and of two major GABA(A)-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the alpha1 and gamma2 subunits of the GABA(A) receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA(A)-receptor subtypes in the postsynaptic membrane.

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A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

Cited by 331 publications

References 31 publications

Eric A. Barnard. 2 July 1927—23 May 2018

Eric A. Barnard. 2 July 1927—23 May 2018

Studies on the Brain Γ-Aminobutyric Acid/Benzodiazepine Receptor and Its Antibodies

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

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