Search PubMed⌕ Search

Biomedical subjects

F A Stephenson

Publications and source records attributed to F A Stephenson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Biochemical evidence for the existence of a pool of unassembled C2 exon-containing NR1 subunits of the mammalian forebrain NMDA receptor.

Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911-920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911-920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17-35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a co-association of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911-920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.

Age Factors↗
PubMed1997

Molecular dissection of native mammalian forebrain NMDA receptors containing the NR1 C2 exon: direct demonstration of NMDA receptors comprising NR1, NR2A, and NR2B subunits within the same complex.

The subunit compositions of the NR1 C2 exon-containing N-methyl-D-aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.

Alternative Splicing↗
PubMed1997

Regulation of neuronal and recombinant GABA(A) receptor ion channels by xenovulene A, a natural product isolated from Acremonium strictum.

Xenovulene A (XR368) is a natural product exhibiting little structural resemblance with classical benzodiazepines yet is able to displace high-affinity ligand binding to the benzodiazepine site of the gamma-aminobutyric acid (GABA)A receptor. We have characterized this compound and an associated congener (XR7009) by use of radioligand binding and electrophysiological methodologies with native neurons and the Xenopus oocyte expression system. Xenovulene A, and the more potent XR7009, inhibited [3H]flunitrazepam binding to rat forebrain with Ki values of 7 and 192 nM, and 1.7 and 42 nM, respectively, each site accounting for approximately 50% of the total specific binding. In cerebellar and spinal cord membranes, these ligands identified only single binding sites. These ligands demonstrated no intrinsic agonist activity at recombinant GABA(A) receptors comprising alpha1beta1gamma2S subunits expressed in Xenopus oocytes, yet at 1 microM both significantly potentiated the GABA-induced response and reduced the GABA EC50 from 10.9 (control) to 5.1 (Xenovulene A) or 2.7 microM (XR7009). The rank potency order for enhancement of the 10 microM GABA response is: XR7009 (EC50, 0.02 microM) > diazepam (0.03) > Xenovulene A (0.05) > flurazepam (0.17). The activity of XR368 and XR7009 was reduced by the benzodiazepine antagonist, flumazenil, and absent in receptors devoid of the gamma2 subunit. These agents exhibited receptor subtype selectivity because alpha3beta1gamma2S receptors were less sensitive to these compounds relative to alpha1 subunit-containing receptors, whereas alpha6beta1gamma2S receptors were completely insensitive. Potentiation of the response to GABA on native GABA(A) receptors in cortical neurons substantiates the profile of the novel structures of Xenovulene A and XR7009 as specific benzodiazepine agonists.

Acremonium↗
PubMed1996

Developmental regulation of expression of GABAA receptor alpha 1 and alpha 6 subunits in cultured rat cerebellar granule cells.

We have studied the postnatal development of GABAA receptor alpha 1 and alpha 6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor alpha 1 and alpha 6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-alpha 1 subunit-specific antibodies, immunoreactive species with M(r) 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the alpha 1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da alpha 1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3-5 DIV in P2 cultures. The GABAA receptor alpha 6 subunit-like immunoreactivity (58,000 Da) was not detected until 5-7 DIV in P10 and 9-11 DIV in P2-derived cultures. The appearance of alpha 6 subunit-like immunoreactivity was paralleled by an up-regulation of alpha 1 subunit expression and a concomitant increase in diazepam-insensitive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of alpha 6 and alpha 1 alpha 6-subunit-containing GABAA receptors (Pollard et al. J. Biol. Chem., 270, 21,285-21,290, (1995)). Antagonism of both non-NMDA and NMDA subtypes of ionotropic glutamate receptors did not significantly affect the developmental profile, the level of GABAA receptor alpha 6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor alpha 6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1996

Bidirectional regulation of GABAA receptor alpha1 and alpha6 subunit expression by a cyclic AMP-mediated signalling mechanism in cerebellar granule cells in primary culture.

Forskolin treatment of cerebellar granule cells in culture resulted in bidirectional regulation of the expression of GABAA receptor alpha1 and alpha6 subunits. Thus, forskolin applied at 2 days in vitro (DIV) increased expression of the alpha1 subunit but decreased the expression of the alpha6 subunit. Values with respect to control cultures, both assayed at 9 DIV by immunoblotting, were 310 +/- 48% for alpha1 and 25 +/- 16% for the alpha6 subunit. Similar effects were evoked following chronic treatment with both dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine. Dideoxyforskolin had no effect on the level of expression of either the alpha1 or the alpha6 GABAA receptor subunits. The changes in subunit expression were accompanied by a 1.7-fold increase in number of total specific [3H]Ro 15-4513 binding sites expressed by intact cerebellar granule cells. This increase in total binding sites was accommodated by a 2.7-fold increase in number of diazepam-sensitive Ro 15-4513 binding sites in accordance with the observed increase in alpha1 subunit expression. The number of diazepam-insensitive subtype of binding sites were not significantly changed. These results suggest that GABAA receptor subtype expression can be differentially regulated by intracellular cyclic AMP concentration.

1-Methyl-3-isobutylxanthine↗
PubMed1996

The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells.

Although three distinct subunits seem to be sufficient to form a functional pentameric GABAA receptor channel, cerebellar granule cells express nRNA for nine subunits. They receive GABAergic input from a relatively homogenous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of granule cells or whether some of them have differential subcellular distribution resulting in distinct types of synaptic and/or extrasynaptic channels. Antibodies to different parts of the alpha 6 and alpha 1 subunits of the GABAA receptor and electron microscopic immunogold localization were used to determine the precise subcellular distribution of these subunits in relation to specific synaptic inputs. Both subunits were present in the extrasynaptic dendritic and somatic membranes at lower densities than in synaptic junctions. The alpha 6 and alpha 1 subunits were colocalized in many GABAergic Golgi synapses, demonstrating that both subunits are involved in synaptic transmission in the same synapse. Synapses immunopositive for only one of the alpha subunits were also found. The alpha 6, but not the alpha 1, subunit was also concentrated in glutamatergic mossy fiber synapses, indicating that the alpha 6 subunit may have several roles depending on its different locations. The results demonstrate a partially differential synaptic targeting of two distinct GABAA receptor subunits on the surface of the same type of neuron.

Animals↗
PubMed1995

Quantitative characterization of alpha 6 and alpha 1 alpha 6 subunit-containing native gamma-aminobutyric acidA receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer.

gamma-Aminobutyric acidA (GABAA) receptors were purified from adult rat cerebella by anti-alpha 6(1-16 Cys) antibody affinity chromatography. Immunoblots of the alpha 6 subunit-containing receptors showed the copurification of the alpha 1, beta 2/3, gamma 2, delta but not alpha 2 and alpha 3 GABAA receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABAA receptor subunit alpha 6(1-16 Cys) and anti-alpha 1(413-429) antibody affinity columns in series substantiated the coassociation of the alpha 1 and alpha 6 polypeptides. The percentage of coexistence of the two subunits was determined by quantitative immunoblotting, which found that 41 +/- 12% of alpha 6 subunit immunoreactivity is associated with the alpha 1 subunit. The ratios of the alpha 1:alpha 6 subunits in the double purified receptor preparations was found to be 1:1, thus determining directly for the first time subunit ratios within native GABAA receptors. The benzodiazepine pharmacology of the alpha 1 alpha 6 subunit-containing receptors was shown to be predominantly benzodiazepine-insensitive by quantitative immunoprecipitation assays. These results are the first direct quantitative studies of subunit ratios within a population of native GABAA receptors.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule.

The N-methyl-D-aspartate R1 (NMDA R1), NMDA R2A, and NMDA R2C subunits were expressed transiently in double or triple combinations in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult mouse forebrain and cerebellum. Under conditions established for maximal expression, cotransfection of the NMDA R1 and R2C subunits yielded a protein detected immunologically with a molecular size of 780,000-850,000 daltons. No cell death was observed in the transfected cells, and the KD for [3H]MK801 binding to the NMDA R1/R2C receptor was 346 +/- 158 nM. This was in contrast to a value of KD = 22 +/- 9 nM found for native cerebellar receptors. Co-transfection with NMDA R1/R2A/R2C subunits with a DNA ratio, 1:3:3, resulted in the expression of a protein with a size similar to the NMDA R1/R2C combination, but the affinity of [3H]MK801 was now 22 +/- 5 nM, and the percentage cell death post-transfection was 89 +/- 17%. Immunoprecipitation assays of detergent-solubilized transfected cells with NMDA R1 subunit-specific antibodies co-precipitated the NMDA R2A and NMDA R2C subunits in 1/2A and 1/2C transfections, respectively. Similarly, immunoprecipitations with either NMDA R1 or NMDA R2C subunit-specific antibodies co-precipitated the NMDA R2A subunit in the R1/2A/2C triple transfections. These results show that the three NMDA receptor subunit types can co-assemble following their co-expression in mammalian cells with a pharmacological profile that is similar to that found for adult cerebellar NMDA receptors.

Amino Acid Sequence↗
PubMed1994

Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes.

1. Human embryonic kidney (HEK) 293 cells were transiently transfected with cDNAs encoding the NR1a-NMDA epsilon 1[NR2A] subunit combination of the NMDA receptor. Single channel behaviour was recorded from outside-out membrane patches, with the aim of comparing the results with those, recorded under the same conditions, from Xenopus oocytes injected with messenger RNA coding for the NR1a-NR2A combination. 2. Single channels in HEK 293 cells showed a main conductance level of 51.4 +/- 2.4 pS, compared with 50.1 +/- 1.4 pS for channels in oocytes. A subconductance level of 38.1 +/- 2.1 pS was found in HEK 293 cells, compared with 38.3 +/- 1.3 pS in oocytes. The frequencies of transitions between the shut and the two conductance levels were also very similar. 3. Distributions of shut times could be fitted with five exponential components. In HEK 293 cells the first three of these components had time constants of 39 +/- 4 microseconds, 0.54 +/- 0.04 ms and 9.94 +/- 1.3 ms; in oocytes the values were 69 +/- 35 microseconds, 0.54 +/- 0.15 ms and 6.53 +/- 4.6 ms, respectively. The relative areas of the components were also similar in the two systems. 4. The distribution of all apparent open times for the sublevels was fitted with two exponential components giving time constants of 0.18 +/- 0.02 ms and 1.31 +/- 0.17 ms (for HEK cells) or of 0.31 +/- 0.36 ms and 1.31 +/- 1.1 ms (for oocytes).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Identification of GABAA receptor subunits expressed in bovine adrenal medulla.

The GABAA receptor subunits expressed in adrenal medulla have been characterized by radioligand binding and by immunological methods. The receptors were purified by both benzodiazepine affinity chromatography and anti-alpha 1 413-429 GABAA receptor antibody immunoaffinity chromatography. These preparations were screened by immunoblotting using GABA receptor alpha subunit sequence-specific antibodies. Results showed the existence of the alpha 1 subunit. Thus, the GABAA receptors expressed in adrenal medulla are homogeneous with respect to their alpha subunit complement and consistent with type BZ1 benzodiazepine receptor pharmacology.

Adrenal Medulla↗
PubMed1994

Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality.

Transient expression of wild-type N-methyl-D-aspartate, NMDAR1-1a/NMDAR2A heteromeric receptors, in mammalian cells yields cell death which was prevented by the inclusion of NMDA receptor antagonists in the cell culture media post-transfection. Transient expression of mutant NMDAR1-1a (N598Q)/NMDAR2A receptors resulted in a significant decrease in the percentage of cell death post-transfection. This mutation has been shown to reduce the Ca2+ permeability of cloned NMDA receptors. Thus these results provide indirect evidence for cell death via an NMDA receptor, Ca(2+)-mediated mechanism.

2-Amino-5-phosphonovalerate↗
PubMed1994

GABAA receptor subtypes expressed in cerebellar granule cells: a developmental study.

The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABAA receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABAA receptor alpha 1 subunit was detectable in immunoblots and increased in level up to day 9. The GABAA receptor alpha 6 subunit was not detectable at day 1; however, at days 3-5, a specific M(r) 58,000 anti-alpha 6 1-16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABAA receptor alpha 6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [3H]Ro 15-4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [3H]Ro 15-4513 sites was found. This was concomitant with the appearance of the GABAA receptor alpha 6 subunit. These results are in agreement with the pharmacology described for alpha 6 beta gamma 2 cloned receptors. They suggest a developmentally regulated expression of the GABAA receptor alpha 6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.

Aging↗
PubMed1993

Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Further evidence for the existence of alpha subunit heterogeneity within discrete gamma-aminobutyric acidA receptor subpopulations.

The alpha subunit complements of natural gamma-aminobutyric acidA (GABAA) receptor subpopulations were investigated by their purification from mammalian cerebral cortex and cerebellum by immunoaffinity chromatography using antibodies raised against peptide sequences unique to the alpha 1, alpha 2, alpha 3, and alpha 6 subunits. Receptors purified from cerebral cortex by anti-Cys alpha 2 414-424 and anti-Cys alpha 3 454-467 antibody affinity columns in series had immunoreactivity with alpha 2 and alpha 3 but not alpha 1 subunit-specific antibodies. Receptors purified from cerebellum by a new affinity column matrix, anti-alpha 6 1-16 Cys whole antibody, or the Fab fragment thereof, enriched for alpha 6 subunit immunoreactivity. A further series of experiments demonstrated the partial coexistence of this alpha 6 subunit immunoreactivity with that for the alpha 1 but not the alpha 2 and alpha 3 subunits. These results provide additional evidence for the existence of GABAA receptor subpopulations with heterogeneous alpha subunit complements yielding increased structural diversity of natural GABAA receptors. Furthermore, they substantiate previous findings implicating the presence of two alpha subunits per receptor oligomer.

Affinity Labels↗