Using a paging system in the rehabilitation of encephalitic patients.

Memory and executive problems following encephalitis are common yet there are few published papers on the successful rehabilitation of such patients. We recently demonstrated (Wilson, Emslie, Quirk, & Evans, 2001; Wilson, Emslie, Quirk, Evans, & Watson, 2005) that a paging system could reduce the everyday memory and planning problems for people with non-progressive brain injury. Among the 143 patients who participated in the 2001 study were four people who had survived encephalitis. Their results are reported here. During a 2-week baseline, the successful task achievement of our four clients ranged from 2-81%. They then received a pager for 7 weeks and task achievement was documented in weeks 6 and 7. All were significantly more successful with the pager than they had been at baseline with success rates ranging from 45-96%. Five weeks after returning their pagers they were monitored once more. One of the encephalitic patients failed to achieve any of his target tasks, returning to baseline level, the other three dropped back a little but were still significantly more successful than at baseline. It is concluded that the paging system can reduce everyday memory and planning problems of patients with encephalitis.

Continuous administration of low-dose GnRH in mares I. Control of persistent anovulation during the ovulatory season.

Three experiments were conducted during the operational breeding season to confirm that continuous, subcutaneous infusion of low-dose GnRH would not disrupt established estrous cycles (Experiment 1), and test the hypotheses that a similar treatment would stimulate secretion of LH and induce development of ovulatory follicles in persistently anovulatory mares (Experiments 2 and 3). Treatment with GnRH (5 microg/h) increased (P<0.001) serum P4 during the luteal phase (7.7+/-0.5 versus 6.4+/-0.5 ng/mL), tended to increase serum LH (2.6+/-0.27 versus 1.9+/-0.25 ng/mL), and did not modify interovulatory intervals. In Experiment 2, GnRH treatment (2.5-5 microg/h) of persistently anovulatory mares increased (P<0.001) serum LH compared to controls (0.5+/-0.08 versus 0.1+/-0.03 ng/mL), with all GnRH-treated and no Control mares ovulating. Mares exhibiting Delayed Recrudescence (n=29) or Lactational Anovulation (n=18), were assigned randomly in Experiment 3 to receive either (1) GnRH/GnRH (n=23); 2.5 microg GnRH/h for 14 d (Period I) and 5 microg/h during the subsequent 28 d (Periods II and III); or (2) Control/GnRH (n=24); no treatment during Period I (control period) and GnRH treatments as in 1 during Periods II and III. Percentage of mares ovulating and pregnant during Period I was greater (P<0.05) for GnRH-treated than Control mares. Thereafter, cumulative ovulation frequency (85%), pregnancy (72%) and cycles/conception (1.3+/-0.2) were similar between groups; however, interval to conception was reduced (P<0.01) by 10.3 d in GnRH/GnRH compared to Control/GnRH.

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation.

BACKGROUND AND PURPOSE: The aim of this report is to study mechanisms of G protein activation by agonists. EXPERIMENTAL APPROACH: The association and dissociation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D(2short) receptor was studied in the presence of a range of agonists. KEY RESULTS: Binding of [(35)S]GTPgammaS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D(2) receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [(35)S]GTPgammaS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [(35)S]GTPgammaS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [(35)S]GTPgammaS binding was reduced with longer association times and increased [(35)S]GTPgammaS concentrations. CONCLUSIONS AND IMPLICATIONS: These data are consistent with [(35)S]GTPgammaS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [(35)S]GTPgammaS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Indoline derivatives as 5-HT(2C) receptor agonists.

A series of 1-(1-indolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for all of the compounds at 5-HT(2) receptor subtypes are reported. A number of compounds were found to reduce food intake in rats after oral administration.

Solubilisation and immunoprecipitation of rat striatal adenosine A(2A) receptors.

In the present study, we have sought to solubilise adenosine A(2A) receptors from rat striatal membranes using a variety of different detergents. Of the detergents tested, 1% CHAPS (3-[(3-deoxycholic acid (cholamidopropyl) dimethylammonio]-1-propanesulfonate) yielded optimal conditions for solubilisation (in the presence of 3 mg/ml protein, 44% of receptor was solubilised, 50% of total protein was solubilised). An antipeptide antibody was raised against a 15 amino-acid sequence within the predicted third intracellular loop region of the human and rat adenosine A(2A) receptor. The antibody was coupled to protein A immobilised on sepharose CL-4B and used to immunoprecipitate adenosine A(2A) receptors from solubilised rat striatal preparations. Radioligand-binding studies were performed using the selective adenosine A(2) antagonist [(3)H]ZM 241385 (4-(2-[7-amino-2-(2-fury1)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). Using [(3)H]ZM 241385, the pharmacology of immunoprecipitated adenosine A(2A) receptors was composed to striatal membrane bound adenosine A(2A) receptors and detergent solubilised adenosine A(2A) receptors. [(3)H]ZM 241385 labelled a single saturable binding site with high affinity in all three preparations (membrane bound K(d) 2.7 nM+/-1.0; solubilised K(d) 1.9 nM+/-0.3; immunoprecipitated K(d) 2.2 nM+/-0.7). Additionally, all three assays confirmed a rank order of potency for displacers consistent with adenosine A(2A) receptor pharmacology: ZM 241385>KW 6002 ((E)-8-[2-(3,4-dimethoxyphenyl)ethynyl]-1-3-diethyl-3,7-dihydro-7-methyl-1-purine 2,6 dione)>CGS 21680, (2-(4-(2 carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosine)>DPCPX (8-cyclopentyl-1,3-dipropylxanthine). We conclude that we have solubilised and immunoprecipitated adenosine A(2A) receptors from rat striatum and that their pharmacology is consistent with native striatal adenosine A(2A) receptors.

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.

Reducing everyday memory and planning problems by means of a paging system: a randomised control crossover study.

OBJECTIVES: To evaluate a paging system designed to improve independence in people with memory problems and executive deficits. METHODS: After a successful pilot study, a randomised control trial was conducted involving a crossover design with 143 people aged between 8 and 83 years. All had one or more of the following: memory, planning, attention, or organisation problems. Most had sustained a traumatic head injury or a stroke although a few had developmental learning difficulties or other conditions. The crossover design ensured that some people received a pager after a 2 week baseline whereas others were required to wait for 7 weeks after the baseline before receiving the pager. Participants were assessed at three time periods-namely, at baseline, 7 weeks, and at 14 weeks postbaseline. RESULTS: More than 80% of those who completed the 16 week trial were significantly more successful in carrying out everyday activities (such as self care, self medication, and keeping appointments) when using the pager in comparison with the baseline period. For most of these, significant improvement was maintained when they were monitored 7 weeks after returning the pager. CONCLUSIONS: This particular paging system significantly reduces everyday failures of memory and planning in people with brain injury.

N-(indol-3-ylglyoxylyl)piperidines: high affinity agonists of human GABA-A receptors containing the alpha1 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.

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics.

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.

Photoaffinity labeling of the benzodiazepine binding site of alpha1beta3gamma2 gamma-aminobutyric acidA receptors with flunitrazepam identifies a subset of ligands that interact directly with His102 of the alpha subunit and predicts orientation of these within the benzodiazepine pharmacophore.

Photoincorporation of ligands into the benzodiazepine site of native gamma-aminobutyric acidA (GABAA) receptors provides useful information about the nature of the benzodiazepine (BZ) binding site. Photoincorporation of flunitrazepam into a single population of GABAA receptors, recombinant human alpha1beta3gamma2, was investigated to probe further the mechanism and orientation of flunitrazepam and other ligands in the BZ binding site. It was concluded that the receptor is primarily derivatized with the entire, unfragmented, flunitrazepam molecule, which undergoes a conformational change during photolysis and largely vacates the benzodiazepine binding site. Investigation of the BZ site after photoincorporation of [3H]flunitrazepam confirmed that binding of other radioligands was unaffected by incorporation of flunitrazepam. This did not correlate with their efficacy but depended on the presence of particular structural features in the molecule. It was observed that affected compounds have a pendant phenyl moiety, analogous to the 5-phenyl group of flunitrazepam, which are proposed to overlap and interact with the same residue or residues in the BZ binding site. Because the major site of flunitrazepam photoincorporation has been shown to be His102, we propose that this group of compounds interacts directly with His 102, whereas compounds of other structural types have no direct interaction with this amino acid. The orientation of ligands within the BZ binding site and their specific interaction with identified amino acids are not well understood. The data in the current study indicate that His102 interacts directly with the pendant phenyl group of diazepam, and further implications for the pharmacophore of the BZ binding site are discussed.

Anticonvulsant activity of azirino[1,2-d][1,4]benzodiazepines and related 1,4-benzodiazepines in mice.

The anticonvulsant properties of several 1,4-benzodiazepine and azirino[1,2-d][1,4]benzodiazepine (ABDZ) derivatives were studied after intraperitoneal (IP) administration in DBA/2 mice (a strain genetically susceptible to sound-induced seizures) and in Swiss mice. The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a hemispheric Perspex dome or on seizures induced by administration of pentylenetetrazole. The 1,4-benzodiazepines were generally more potent than the related ABDZ derivatives. The rank order of potency for anticonvulsant activity was flunitrazepam > diazepam > pinazepam > ABDZ5 > ABDZ4 > prazepam > halazepam > ABDZ1 > ABDZ3 > camazepam > ABDZ6 > ABDZ2. The impairment of locomotor performance following IP administration of these derivatives was also evaluated by means of the rotarod test. The rank order of potency for impairment of coordinated motor movements was pinazepam > flunitrazepam > diazepam > ABDZ5 > prazepam > halazepam > ABDZ4 > ABDZ3 > ABDZ1 > camazepam > ABDZ2 = ABDZ6. The potency of various 1,4-benzodiazepines and ABDZs as inhibitors of specific [3H]flumazenil binding to membranes from cerebellum or cortex was evaluated. In general, ABDZs were active as anticonvulsants and inhibited [3H]flumazenil binding in the micromolar range. Radioligand binding studies carried out in stable cell lines demonstrated that none of the ABDZs tested showed a particular subtype specificity. The pharmacological actions of ABDZ4 and ABDZ5, which appeared to be the most potent ABDZs as anticonvulsants, were significantly reduced by treatment with flumazenil (8.24 mumol/kg IP), suggesting a clear involvement of benzodiazepine mechanisms in the anticonvulsant activity of these compounds or their metabolites. The anticonvulsant activity of ABDZ4 and ABDZ5 was also evaluated against seizures induced in DBA/2 mice by two beta-carbolines: methyl-beta-carboline-3-carboxylate (beta-CCM) and methyl-6,6-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). Both ABDZ4 and ABDZ5 give better protection against seizures induced by beta-CCM than DMCM, suggesting a preferential action on the benzodiazepine receptor subtype BDZ1.

[3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit.

A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABAA receptors containing an alpha 5 subunit over those containing an alpha 1, alpha 2, alpha 3 or alpha 6 subunit in combination with beta 3 and gamma 2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit. [3H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a Kd of 2.4 +/- 0.7 nM and a Bmax of 256 +/- 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with alpha 5, beta 2 and gamma 2 and with receptors immunoprecipitated from rat brain with an alpha 5-selective antiserum. It is concluded that [3H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABAA receptor and should provide a valuable tool for elucidating the structure and function of the alpha 5-containing GABAA receptor subtype.

Characterisation of delta-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.

Effects of L-690,488, a prodrug of the bisphosphonate inositol monophosphatase inhibitor L-690,330, on phosphatidylinositol cycle markers.

In order to enhance the entry into cells of L-690,330, a bisphosphonate inhibitor of inositol monophosphatase (IMPase; a key, enzyme in the phosphatidylinositol (Pl) cell signaling pathway), the tetrapivaloyloxymethyl ester prodrug, L-690,488 [tetrapivaloyloxymethyl 1-(4-hydroxyphenoxy)ethane-1,1-bisphosphonate], was synthesized. The effects of L-690,488 were studied in cholinergically (carbachol)-stimulated rat cortical slices and Chinese hamster ovary cells stably transfected with the human muscarinic m1 receptor (m1 CHO cells). The accumulation of [3H]inositol monophosphates or [3H]cytidine monophosphorylphosphatidate ([3H]CMP-PA) after [3H]inositol or [3H]cytidine prelabeling, respectively, and inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate mass were measured. In rat cortical slices and m1 CHO cells, the maximum response and time course of accumulation of [3H]inositol monophosphates for L-690,488 and lithium were similar. However, the concentrations of L-690,488 required to produce these effects (EC50 values of 3.7 +/- 0.9 and 1.0 +/- 0.2 microM in cortical slices and m1 CHO cells, respectively) were much lower than with lithium (0.3-1.5 mM). Likewise, the time course and maximum accumulation of [3H] CMP-PA in L-690,488-treated m1 CHO cells was similar to lithium but L-690,488 was again much more potent (EC50 values = 3.5 +/- 0.3 microM and 0.52 +/- 0.03 mM for L-690,488 and lithium, respectively). In addition, L-690,488 attenuated the carbachol-induced elevation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in m1 CHO cells, an effect reported previously with lithium. These results are all consistent with L-690,488 and lithium both depleting intracellular inositol as a consequence of inhibition of IMPase. That these effects of L-690,488 on the PI cycle are indeed due to inositol depletion is shown by the observation that the effects of L-690,488 on CMP-PA accumulation could be overcome by addition of exogenous myo-inositol (EC50 = 1.7 +/- 0.5 mM). These data show that inhibition of IMPase produces effects on the PI cycle comparable to lithium. As a corollary, the effects of lithium on the PI cycle are therefore consistent with its major mechanism of action being inhibition of IMPase.

gamma-Aminobutyric acid type A receptors in the rat brain can contain both gamma 2 and gamma 3 subunits, but gamma 1 does not exist in combination with another gamma subunit.

Antibodies specific for the gamma 1, gamma 2, and gamma 3 subunits of the gamma-aminobutyric acid (GABA)A receptor have been used to probe the composition of naturally occurring GABAA receptors in the rat brain. Most GABAA receptors contain at least one of these three subunits. The percentage of each, determined by immunoprecipitation of [3H]muscimol binding, was 11 +/- 1%, 59 +/- 3%, and 14 +/- 2% for gamma 1, gamma 2, and gamma 3 subunits, respectively. Receptors containing gamma 2 or gamma 3 subunits were labeled by benzodiazepine site ligands with high affinity, whereas gamma 1-containing receptors could be labeled only by [3H]muscimol. Receptors immunoprecipitated by anti-gamma 2 or anti-gamma 3 antibodies were labeled with [3H]Ro 15-1788 with similar affinities (Kd for anti-gamma 2-immunoprecipitated receptors, 1.9 nM; Kd for anti-gamma 3-immunoprecipitated receptors, 1.7 nM). Immunoprecipitation or Western blot analysis of GABAA receptors solubilized from rat cerebellar or whole-brain preparations indicated that gamma 1 was not present coassembled with any other gamma subunit. Western blot analysis of receptors purified on alpha-specific immunoaffinity resins showed that gamma 1 was predominantly assembled with the alpha 2 subunit. Some GABAA receptors may contain more than one type of gamma subunit. Quantitative immunoprecipitation and Western blot analysis both indicated that gamma 2 and gamma 3 subunits can exist in the same receptor complex. A large proportion of GABAA receptors immunopurified on a gamma 3 affinity resin also appeared to contain a gamma 2 subunit. In contrast, when receptors were purified on a gamma 2 affinity resin a small proportion also appeared to contain a gamma 3 subunit. We conclude that most gamma 1-containing receptors have no other gamma subunit in the same receptor complex but some GABAA receptors contain both gamma 2 and gamma 3 subunits.

Model of subunit composition of gamma-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their alpha and gamma/delta subunits.

Antibodies specific for subunits of the gamma-aminobutyric acid A (GABAA) receptor have been used to immunoprecipitate [3H]muscimol, [3H]Ro 15-4513, and [3H]Ro 15-1788 binding sites from deoxycholate-solubilized preparations of rat cerebellum. Of the antisera raised against alpha subunits, those specific for alpha 6 immunoprecipitated the largest proportion of receptors. Two populations of alpha 6-containing GABAA receptors were identified. The first was labeled with [3H]Ro 15-4513 and exhibited a pharmacological profile consistent with that observed for alpha 6 beta 2 gamma 2 in transfected cells (Lüddens, H., Pritchett, D. B., Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeberg, P. H. (1990) Nature 346, 648-651). The second population was labeled only with [3H]muscimol and was deduced, from quantitative immunoprecipitation studies using combinations of antibodies, to contain both alpha 6 and delta subunits. The alpha 6 subunit was not observed to be present in combination with other alpha subunits or the gamma 1 subunit. Each of the other alpha subunits was found to be present in only one population of receptors in the cerebellum. Some subunits (alpha 4, alpha 5, and gamma 3) were not detectable. By combining information from quantitative immunoprecipitation experiments and Western blot analysis, a model describing the composition of all GABAA receptors in the cerebellum was constructed that defined the following alpha and gamma/delta combinations (percentage of cerebellar GABAA receptors): alpha 6 gamma 2 (36%), alpha 6 delta (23%), alpha 1 gamma 2 (28%), alpha 2 gamma 1 (8%), and alpha 3 gamma 2 (5%).

A novel allosteric modulatory site on the GABAA receptor beta subunit.

Cloning of cDNAs that code for GABAA receptor subunits has revealed multiple receptor populations constructed from different subunit combinations. On native rat and cloned human GABAA receptors, the anticonvulsant compound loreclezole strongly potentiated GABA-mediated chloride currents. Using different combinations of human GABAA receptor subunits expressed in Xenopus oocytes and transfected 293 cells, loreclezole was highly selective for receptors containing the beta 2 or beta 3 subunit over those containing the beta 1 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 GABAA receptor beta subunit that allows pharmacological discrimination of different GABAA receptor subpopulations in the brain and provides a new target for putative anticonvulsant/anxiolytic drugs.

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.