Kathleen Quirk

GABAA receptor subtypes immunopurified from rat brain with α subunit-specific antibodies have unique pharmacological properties

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.

A novel allosteric modulatory site on the GABAA receptor β subunit

Abstract Cloning of cDNAs that code for GABA A receptor subunits has revealed multiple receptor populations constructed from different subunit combinations. On native rat and cloned human GABA A receptors, the anticonvulsant compound loreclezole strongly potentiated GABA-mediated chloride currents. Using different combinations of human GABA A receptor subunits expressed in Xenopus oocytes and transfected 293 cells, loreclezole was highly selective for receptors containing the β2 or β3 subunit over those containing the β1 subunit. Loreclezole was demonstrated to act at a site distinct from the benzodiazepine, barbiturate, and steroid sites with a unique subunit dependence. These results describe a previously unidentified modulatory site on the GABA A receptor β subunit that allows pharmacological discrimination of different GABA A receptor subpopulations in the brain and provides a new target for putative anticonvulsant/anxiolytic drugs.

Characterisation of δ-subunit containing GABAA receptors from rat brain

Polyclonal antibodies have been raised in rabbits against the predicted cytoplasmic loop region of the delta-subunit of the GABAA receptor. These specifically identify the expressed fragment by Western blot but do not cross react with analogous polypeptides from the gamma 1, gamma 2 or gamma 3-subunits. Polyclonal antisera immunoprecipitated [3H]muscimol binding sites from several brain regions consistent with the reported distribution of delta-subunit mRNA and also detected the delta-subunit by Western blot, identifying a polypeptide of 55KDa. Receptors immunoprecipitated from rat brain with the delta-antisera exhibited an atypical profile with respect to their radioligand binding properties. Receptors immunoprecipitated from all regions tested bound [3H]muscimol, but did not bind benzodiazepine site ligands [3H]Ro 15,1788 or [3H]flunitrazepam with high affinity. Receptors containing a delta-subunit accounted for 10.7 +/- 2% of all GABAA receptors ([3H]muscimol binding sites) in the rat central nervous system as deduced from quantitative immunoprecipitation experiments, the largest population being in the cerebellum where approximately 27% of all receptors contained a delta-subunit. The pharmacology of the GABA (gamma-aminobutyric acid) binding site on receptors immunoprecipitated from cerebellum with gamma 2 and delta-antisera was compared. The rank order of potency of a series of 6 compounds to compete for [3H]muscimol binding sites was similar in these two populations, but muscimol had a significantly higher affinity for receptors containing the delta-subunit. These receptors therefore comprise a novel population of GABAA receptors which do not bind benzodiazepines but have a 5-fold higher affinity for muscimol.(ABSTRACT TRUNCATED AT 250 WORDS)

THE PHARMACOLOGY OF THE BENZODIAZEPINE SITE OF THE GABA-A RECEPTOR IS DEPENDENT ON THE TYPE OF GAMMA -SUBUNIT PRESENT

The pharmacology of native and recombinant GABA-A receptors containing either gamma1, gamma2 or gamma3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual gamma-subunits and analysing their radioligand binding characteristics. Receptors containing a gamma1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a gamma2 or gamma3 subunit bind [3H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of alpha-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition alpha1beta3gamma2 followed by immunoprecipitation of individual subunits revealed that the alpha and gamma but not the beta-subunit could be irreversibly labelled by [3H]flunitrazepam. The properties of recombinant receptors have been investigated in oocytes expressing gamma1, gamma2, or gamma3 subunits in combination with an alpha and a beta-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different gamma-subunits. Others such as CL 218,872 show no selectivity between receptors containing different gamma-subunits but exhibit selectivity for receptors containing different alpha-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the alpha and gamma-subunits.

Characterisation of agonist binding on human 5-HT2C receptor isoforms

The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.

Solubilisation of the 5‐Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

Abstract: 5‐Hydroxytryptamine3 (5‐HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205–930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested { 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X‐100, and 1% octyl glucoside}, deoxycholate (0.5%) yielded the best solubilisation (54.6 ± 6% of receptor, 70.5 ± 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205–930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G‐25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205–930 to these soluble sites was saturable and specific (Bmax, = 46.1 ± 6 fmol/mg of protein; KD= 0.33 ± 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205–930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4°C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists competed for the soluble receptors with a rank order of potency typical for binding at a 5‐HT3 receptor site: zacopride (Ki= 0.26 nM) > quipazine (0.37 nM) = Q ICS 205–930 (0.33 nM) > ICS 205–930 (0.93 nM) > GR 38032F (2.2 nM) > BRL 24924 (4.1 nM) > MDL 72222 (23.4 nM) > ketanserin (6,000 nM). The agonists 5‐HT and 2‐methyl‐5‐HT also competed for [3H]Q ICS 205–930 binding with high affinity (39.6 and 55.6 nM respectively). Therefore, we conclude that the 5‐HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.

N-(Indol-3-ylglyoxylyl)piperidines: high affinity agonists of human GABA-A receptors containing the α1 subunit

A new class of N-(indol-3-ylglyoxylyl)piperidines are high affinity agonists at the benzodiazepine binding site of human GABA-A receptor ion-channels, with modest selectivity for receptors containing the alpha1 subunit over alpha2 and alpha3. All three receptor subtypes discriminate substantially between the two enantiomers of the chiral ligand 10.

Characterization of the solubilized NMDA receptor complex from rat brain with [3H]L-689,560, a glycine site antagonist

An antagonist ligand for the glycine site of the NMDA receptor, [3H]L-689,560, has recently been described. We have investigated the use of this ligand to label NMDA receptors which have been solubilized from rat brain. It has significant advantages over [3H]dizocilpine ([3H]MK-801) for this purpose since (a) it is not inhibited by most detergents, (b) interactions between the glutamate and glycine sites are maintained, and (c) equilibrium binding is rapid and of high affinity (Kd = 8.8 +/- 1.9 nM, n = 4). Nevertheless, precautions must be taken to remove glycine throughout all experimental procedures. In addition we have investigated the ability of NMDA receptors to bind to various lectins and conclude that only N-linked glycosylation is present, consistent with consensus sequences for glycosylation present in cloned subunits of the NMDA receptor. Further binding of the radioligand [3H]L-689,560 was detected both to the solubilized receptor and to receptor immobilized on lectin-agarose, identifying it as an appropriate ligand for use in the characterization of NMDA receptors during purification procedures.

[125I]iodoclonazepam, a specific high affinity radioligand for the identification of BZ1 and BZ2 sites in rat brain.

Binding of the radioligand [125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum Bmax = 1.49 +/- 0.3 pmol/mg of protein, Kd = 0.39 +/- 0.06 nM; hippocampus Bmax = 1.5 +/- 0.14 pmol/mg of protein, Kd = 0.38 +/- 0.6 nM and striatum Bmax = 0.53 +/- 0.1 pmol/mg of protein, Kd = 0.34 +/- 0.03 nm, n = 3). In all regions only one population of sites was apparent. However, competition for [125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The BZ1 selective compounds zolpidem and CL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [125I]iodoclonazepam is a novel, high affinity ligand which recognises both BZ1 and BZ2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.