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F A Stephenson

Publications and source records attributed to F A Stephenson.

At least 19 recordsLinked to original sources
PubMed1992

Mapping of GABAA receptor alpha 5 and alpha 6 subunit-like immunoreactivity in rat brain.

The distribution of the alpha 5 and alpha 6 subunits of the GABAA receptor has been mapped in rat brain using affinity-purified antibodies generated against peptide sequences unique to the respective polypeptides. alpha 5 Subunit-like immunoreactivity was of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. alpha 6 Subunit-like immunoreactivity was observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAA receptor heterogeneity in the CNS.

Amino Acid Sequence↗
PubMed1992

Immunological detection of the NMDAR1 glutamate receptor subunit expressed in embryonic kidney 293 cells and in rat brain.

The rat NMDAR1 (N-methyl-D-aspartate receptor) was expressed transiently in human embryonic kidney cells. Transfected cell homogenates showed saturable [3H]MK-801 binding activity that was best fit by a single high-affinity site with a KD of 9 nM and a Bmax of 113 fmol of binding sites/mg of protein. Antibodies raised against the peptide sequence NMDAR1 (929-938) coupled to keyhole limpet haemocyanin specifically recognised a single band with M(r) 117,000 in immunoblots from adult rat brain. In the transfected cells, the antibody recognised two bands: one with M(r) 117,000, which was coincident with that from brain membranes, and one with M(r) 97,000, which was identified as nonglycosylated NMDAR1 subunit. These results identify the NMDAR1 of rat brain and further show that the homooligomer binds MK-801, albeit at low efficiency.

Animals↗
PubMed1992

Quantitative immunoprecipitation studies with anti-gamma-aminobutyric acidA receptor gamma 2 1-15 Cys antibodies.

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.

Amino Acid Sequence↗
PubMed1991

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.

Animals↗
PubMed1991

Promiscuity of GABAA-receptor beta 3 subunits as demonstrated by their presence in alpha 1, alpha 2 and alpha 3 subunit-containing receptor subpopulations.

Polyclonal antibodies were raised in rabbits against the GABAA-receptor beta 3 subunit peptide sequence, KQSMPREGHGRHMDR-NH2 coupled to keyhole limpet haemocyanin. These anti-beta 3 379-393 antibodies immunoprecipitated in a dose-dependent manner specific benzodiazepine agonist binding sites from Na+ deoxycholate extracts of bovine cerebral cortex. In immunoblots, anti-beta 3 379-393 antibodies recognised two species with Mr 59,900 and Mr 57,200 in all preparations tested, which included crude detergent-solubilised, benzodiazepine affinity chromatography-purified receptor, anti-alpha 1 324-341 antibody, anti-Cys alpha 2 414-424 antibody and anti-Cys alpha 3 454-467 antibody immunoaffinity-purified GABAA-receptor subpopulations. These results provide evidence for the ubiquity and promiscuity of the GABAA-receptor beta 3 subunit.

Amino Acid Sequence↗
PubMed1990

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction.

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.

Affinity Labels↗
PubMed1990

Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties.

The cloned cDNAs encoding the alpha 1 and beta 1 subunits of the bovine brain GABA(A) receptor have been co-transfected, using a dexamethasone-inducible promoter, into cultured hamster ovary cells, with selection to form a stable cell line. The use, alternatively, of a much stronger constitutive promoter led to cell death consequent upon high receptor density. After induction, the cells contained the alpha 1 and beta 1 mRNAs. The expressed receptors showed the high-affinity binding of [3H]muscimol and of the GABA(A) receptor channel blocker, t-butylphosphorothionate (TBPS), and the characteristic enhancement of the former by a pregnanolone. Their GABA-activated current was potentiated by the barbiturate, pentobarbitone, was reversibly blocked by bicuculline and picrotoxin, but was not enhanced by benzodiazepines. In mouse spinal cord neurons GABA activates channel openings to at least four conductance states (45, 30, 19 and 12 pS) with the 30 pS state being the most frequently observed (main) state. However, the main state of the alpha 1/beta 1 GABA(A) receptor was the 19 pS state. The enhancement of GABA(A) receptor current by barbiturates wa due to prolongation of mean channel lifetime, whereas the reduction of GABA(A) receptor current by picrotoxin was due to reduction of channel opening frequency and mean channel lifetime. Stable cell lines containing subunit combinations of this receptor should provide a powerful tool for the elucidation of its channel features and control mechanisms.

Animals↗
PubMed1990

Biochemical evidence for the existence of gamma-aminobutyrateA receptor iso-oligomers.

Polyclonal antibodies were raised against synthetic peptides whose sequences were from unique regions of the bovine gamma-aminobutyrateA receptor alpha 1, alpha 2, and alpha 3 subunits. The anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 antibodies all specifically immunoprecipitated [3H]flunitrazepam and [3H]muscimol binding activities in parallel from Na+ deoxycholate extracts of bovine cerebral cortex. The maximum number of benzodiazepine binding sites immunoprecipitated by each antibody in three brain regions, cerebral cortex, cerebellum, and hippocampus, was investigated. Differences were found for both the maximum number of sites immunoprecipitated by each antibody in one brain region and for the percentage of benzodiazepine binding sites immunoprecipitated by one specificity antibody between the different brain regions. Furthermore, it was found that co-immunoprecipitation with either anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 or anti-alpha 1 324-341 and anti-Cys alpha 3 454-467 antibodies resulted in an increase in the percentage of benzodiazepine binding sites immunoprecipitated, the sum of which was equal to the percentages pelleted by the individual antibodies. These results demonstrate for the first time the existence in mammalian brain of gamma-aminobutyrateA receptor alpha subunit iso-oligomers.

Amino Acid Sequence↗
PubMed1990

Thermotropic properties of saturated mixed acyl phosphatidylethanolamines.

The mixed acyl phosphatidylethanolamine (PE) series C(18)C(18)PE, C(18)C(16)PE, C(18)C(14)PE, C(18)C(12)PE, and C(18)C(10)PE has been prepared from the corresponding phosphatidylcholines by phospholipase D mediated transphosphatidylation. The thermotropic behavior of unhydrated and hydrated preparations of these PEs has been investigated by differential scanning calorimetry and 31P NMR spectroscopy. Unhydrated preparations of the PEs undergo crystalline to liquid-crystalline transitions (Tm+h), which correspond to the simultaneous hydration and acyl chain melting of poorly hydrated crystalline samples. Hydrated preparations of the PEs undergo gel to liquid-crystalline transitions (Tm) when scanned immediately subsequent to cooling from temperatures above their respective Tm+hs. Multilamellar bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE pack without significant interdigitation of the phospholipid acyl chains across the bilayer center in the gel phase. C(18)C(10)PE multilamellar preparations exhibit a mixed-interdigitated gel phase packing of the phospholipid acyl chains. Hydrated bilayers of C(18)C(12)PE adopt a mixed-interdigitated gel phase packing at temperatures below 13.9 degrees C. Between 13.9 degrees C and the gel to liquid-crystalline transition temperature of 36.9 degrees C, the C(18)C(12)PE bilayer adopts a noninterdigitated gel phase packing. The metastable behavior of fully hydrated and partially hydrated preparations of the mixed acyl PEs has been investigated. Bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE exhibited little or no tendency toward regeneration of the crystalline phase. In contrast, bilayers of C(18C(12)PE and C(18)C(10)PE exhibited a metastability of the liquid-crystalline phase in the temperature interval between Tm and Tm+h, which can allow for the regeneration of the crystalline phase under certain conditions. Bilayers of C(18)C(12)PE exhibited an additional metastability of the noninterdigitated gel phase.

Calorimetry, Differential Scanning↗
PubMed1989

Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific gamma-aminobutyric acidA-receptor antibodies.

The gamma-aminobutyric acidA (GABAA) receptor purified from adult bovine cerebral cortex was photoaffinity-labelled with the agonist benzodiazepine [3H]flunitrazepam and the radioactivity shown to be coincident with a band with Mr 53,000 that was recognized by three anti-(GABAA receptor alpha 1 subunit sequence)-specific antibodies. Complete and limited CNBr cleavage of the purified photoaffinity-labelled receptor was carried out. The products of this reaction were analysed for radioactivity, for immunoreactivity with anti-[alpha 1-(1-15)-peptide], anti-[alpha 1-(324-341)-peptide] and anti-[alpha 1-(413-429)-peptide] polyclonal antibodies and for carbohydrate by biotinylated concanavalin A lectin overlay. Complete CNBr cleavage gave a radioactive peptide with Mr 10,000-12,000 that was not recognized by the above-mentioned specific antisera. By using the deduced amino acid sequence of the alpha 1 subunit [Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Ramachandran, Reale, Glencorse, Seeburg & Barnard (1987) Nature (London) 328, 221-227], it is proposed that the site of the benzodiazepine-agonist photoaffinity-labelling reaction does not lie within the amino acid sequences alpha 1 1-58 and alpha 1 149-429.

Affinity Labels↗
PubMed1989

Identification of the alpha 3-subunit in the GABAA receptor purified from bovine brain.

Polyclonal antibodies have been raised to synthetic amino acid sequences of the bovine GABAA receptor alpha 1 and alpha 3 subunits. Anti-alpha 1 subunit antibodies recognise a polypeptide of 53 kDa whereas anti-alpha 3 subunit antibodies recognise a polypeptide of 59-60 kDa, in Western blots of GABAA receptor purified from adult bovine cerebral cortex, cerebellum and 12-day calf cerebral cortex.

Animals↗
PubMed1989

Bovine gamma-aminobutyric acidA receptor sequence-specific antibodies: identification of two epitopes which are recognised in both native and denatured gamma-aminobutyric acidA receptors.

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.

Amino Acid Sequence↗
PubMed1989

Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

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.

Animals↗
PubMed1988

Benzodiazepine binding site heterogeneity in the purified GABAA receptor.

The displacement of [3H]flunitrazepam binding activity by ethyl-beta-carboline-3-carboxylate (beta CCE) was studied in both membrane-bound and purified GABAA receptors from adult bovine cerebral cortex, hippocampus and cerebellum. It was found that the best fit for the displacement of benzodiazepine binding in the cerebellar membranes was a single site with IC50 = 0.55 +/- 0.21 nM, whereas the best fit for cortical and hippocampal membranes was a two-site model with respective values of IC50 = 0.2 +/- 0.09 nM (high affinity), IC50 = 21 +/- 6 nM (low affinity) (cortex) and IC50 = 0.25 +/- 0.05 nM, IC50 = 20 +/- 2 nM (hippocampus). These same properties were retained in the purified GABAA receptor from the three brain regions. Thus, we have demonstrated that binding site heterogeneity as defined by the displacement of beta CCE is preserved in purified GABAA receptors and we suggest that this provides evidence for the existence of GABAA receptor isoforms.

Animals↗
PubMed1988

Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers.

Aqueous dispersons of L-alpha-phosphatidylethanolamine (PE) with identical saturated acyl chains are known to exhibit gel-state metastability. It is also known that the metastability in PE becomes more pronounced with decreasing acyl chain-length. In an attempt to study the metastable phase behavior of PE, we have synthesized diundecanoylphosphatidylethanolamine (diC11PE) and examined its polymorphic phase behavior. A single endothermic transition at 38 degrees C is detected between 10 and 55 degrees C by DSC for the nonheated sample of diC11PE in excess water. An immediate second heating scan done after cooling slowly of the same sample from the liquid-crystalline state shows a smaller endothermic transition at a lower temperature, 18 degrees C. However, the high-temperature transition at 38 degrees C can be detected, if the sample which has been heated above 38 degrees C is quench cooled from the liquid-crystalline to a temperature between 18 and 38 degrees C. Furthermore, two endothermic transitions at 18 and 38 degrees C and an exothermic transition at 19 degrees C are recorded for diC11PE after quench supercooling of the sample from the liquid-crystalline state to an appropriate temperature below 10 degrees C. The gel-state metastability of diC11PE can be most appropriately explained in terms of changes in interbilayer headgroup-headgroup interactions. It is suggested that the kinetically trapped supercooled metastable state may be a multilamellar structure with melted acyl chains but with strong interbilayer headgroup-headgroup interactions.

Calorimetry↗