Salicylidene salicylhydrazide, a selective inhibitor of beta 1-containing GABAA receptors.

1. A high-throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of alpha2beta1gamma1 GABA(A) receptors with a maximum inhibition of 56+/-5% and an IC(50) of 32 (23, 45) nm. 2. Evaluation of this compound using patch-clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the beta1 subunit (e.g. maximum inhibition of 68.1+/-2.7% and IC(50) value of 5.3 (4.4, 6.5) nm on alpha2beta1gamma1 receptors). The presence of a beta1 subunit was paramount for the inhibition with changes between alpha1 and alpha2, gamma1 and gamma2, and the presence of a subunit having little effect. 3. On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at alpha1beta1gamma1 receptors (74.3+/-1.4%). SCS displayed no use or voltage dependence, suggesting that it does not bind within the channel region. Concentration - response curves to GABA in the presence of SCS revealed a reduction in the maximum response with no change in the EC(50) or Hill coefficient. In addition, SCS inhibited pentobarbitone-induced currents. 4. Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. 5. SCS did not compete with the known allosteric modulators, picrotoxin, pregnenolone sulphate, dehydroepiandrosterone 3-sulphate, bicuculline, loreclezole or mefenamic acid. Neither was the inhibition by SCS influenced by the benzodiazepine site antagonist flumazenil. 6. In conclusion, SCS is unique in selectively inhibiting GABA(A) receptors containing the beta1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the beta1 subunit and the lack of interaction with a range of GABA(A) receptor modulators suggests that SCS is interacting at a previously unidentified site.

The non-peptidyl fungal metabolite L-783,281 activates TRK neurotrophin receptors.

Neurotrophin binding to the extracellular surface of the Trk family of tyrosine kinase receptors leads to the activation of multiple signalling cascades, culminating in neuroregenerative effects, including neuronal survival and neurite outgrowth. Since neurotrophins themselves are not ideal drug candidates due to their poor pharmacokinetic behaviour and bioavailability, small molecule neurotrophin mimetics may be beneficial in treating a number of neurodegenerative disorders. The present study demonstrates that L-783,281, a non-peptidyl fungal metabolite, is capable of stimulating TrkA, B and C phosphorylation to various extents in CHO cells stably expressing human Trk receptors. L-783,281 also stimulated Trk phosphorylation in a number of rat and human primary neuronal cultures, whereas the highly similar compound, L-767,827, was without effect. Mechanistic studies utilizing transiently transfected PDGF/TrkA and TrkA/PDGF chimeras, demonstrated that L-783,281 is likely to interact with the intracellular domain of the TrkA receptor. Further investigations suggested that L-783,281 was nevertheless able to instigate receptor dimerization by binding in a non-covalent manner. Although the cytotoxicity of the compound was shown to preclude its effects in neuronal survival and neurite outgrowth assays, it is a prototype for a small molecule neurotrophin mimetic that activates Trk by interacting at a site different from the neurotrophin-binding site.

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor.

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes.

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.

Mutation at the putative GABA(A) ion-channel gate reveals changes in allosteric modulation.

We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA(A) beta2 and investigated the actions of a number of GABA(A) agonists, antagonists and modulators on human alpha1beta2deltaL259Sgamma2s compared to wild type alpha1beta2gamma2s GABA(A) receptors, expressed in Xenopus oocytes. The mutation resulted in smaller maximum currents to gamma-aminobutyric acid (GABA) compared to alpha1beta2gamma2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC50 (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S) also displayed a decrease in EC50. In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on alpha1beta2deltaL259Sgamma2s receptors, with different degrees of maximum inhibition. The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC20). Compared to wild type, none of these modulators potentiated the EC20 response of alpha1beta2deltaL259Sgamma2s receptors, however they all directly activated the receptor in the absence of GABA. To conclude, the above mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site.

Residues in transmembrane domains I and II determine gamma-aminobutyric acid type AA receptor subtype-selective antagonism by furosemide.

GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other alpha-subunits. Furosemide exhibits approximately 100-fold selectivity for alpha6-containing receptors over alpha1-containing receptors. By making alpha1/alpha6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha6beta3gamma2s receptors. Within the alpha1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta-subunit selectivity of furosemide to be due to asparagine 265 in the beta2 and beta3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at alpha6beta3gamma2s compared with alpha1beta3gamma2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site.

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.

Generation and characterisation of stable cell lines expressing recombinant human N-methyl-D-aspartate receptor subtypes.

Transfection of mouse L(tk-) cells with human N-methyl-D-aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist D, L-(epsilon)-2-[3H]amino-4-propyl-5-phosphono-3-pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6-5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.

beta-Carboline gamma-aminobutyric acidA receptor inverse agonists modulate gamma-aminobutyric acid via the loreclezole binding site as well as the benzodiazepine site.

The benzodiazepine site on the gamma-aminobutyric acid(A) (GABAA) receptor is the principle site of action for a number of structurally diverse compounds, including the beta-carbolines, many of which bind with high affinity. The apparent reversal of inhibition and potentiation by high concentrations of methyl-6,7-dimethoxy-4-ethyl-beta-carboline (DMCM) and other beta-carbolines has been reported by several groups and is insensitive to the benzodiazepine antagonist Ro 15-1788. By using alpha 6-containing receptors, which have low affinity for benzodiazepines, we observed robust potentiation of GABAA responses by micromolar concentrations of DMCM and other beta-carbolines that is dependent on the beta subunit variant. The beta subunit-dependent potentiation by the anticonvulsant loreclezole is dependent on a single amino acid in the putative transmembrane 2 region. By using single point mutations that discriminate the loreclezole site, we show that potentiation by DMCM is also dependent on the presence of the same amino acid, Asn290, in beta 2 or beta 3 (serine in beta 1), providing evidence that the low affinity site for beta-carboline potentiation is the loreclezole site. The potentiation is independent of the alpha subunit and is more pronounced on alpha 6-containing receptors due to the lack of DMCM inhibition via the benzodiazepine site. In addition, the potentiation observed is competitive with that of loreclezole, and other beta-carbolines, such as ethyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate, act in a similar manner. The finding that beta-carbolines can act via the loreclezole site as well as the benzodiazepine site suggests that a wider variety of compounds may act via this site and shows that compounds can interact with more than one modulatory site on the GABAA receptor.

The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.

Type A gamma-aminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligand-gated ion channels probably formed from the coassembly of different subunits (alpha 1-6, beta 1-3, gamma 1-3, delta) in the arrangement alpha beta gamma or alpha beta delta. The activation of these receptors by GABA can be modulated by a range of compounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the beta subunit of the GABAA receptor. The action of loreclezole depends on the type of beta subunit present in the receptor complex; receptors containing beta 2 or beta 3 subunits have > 300-fold higher affinity for loreclezole than receptors containing a beta 1 subunit. We have used this property to identify the amino acid residue in the beta subunit that determines the subunit selectivity of loreclezole. Chimeric beta 1/beta 2 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha 1 and gamma 2s subunits. The chimera beta 1/beta 2Lys237-Gly334 conferred sensitivity to 1 microM loreclezole. Within this region there are four amino acids that are conserved in beta 2 and beta 3 but differ in beta 1. By mutating single amino acids of the beta 1 subunit to the beta 2/beta 3 equivalent, only the beta 1 mutation of Ser-290-->Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the beta 2 and beta 3 subunits to the beta 1 equivalent (Asn-->Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors containing the mutated beta subunits. Thus, a single amino acid, beta 2 Asn-289 (beta 3 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.

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.

Role of the beta subunit in determining the pharmacology of human gamma-aminobutyric acid type A receptors.

A cDNA encoding the human gamma-aminobutyric acid (GABA)A receptor beta 2 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows only a single amino acid change from the rat sequence (Asn-347 in rat, serine in human). Using polymerase chain reaction amplification of human-specific products from human x rodent somatic cell hybrid DNAs, the gene has been assigned to human chromosome 6. By expressing recombinant human GABAA receptors containing different beta subunits (beta 1, beta 2 or beta 3) in both transfected cells and Xenopus oocytes, we have been able to determine the influence of the beta subunit on the pharmacology of the receptor. For a number of benzodiazepine binding site compounds, a barbiturate, and several neurosteroids, neither the affinity nor the efficacy of the compounds is influenced by the type of beta subunit present in the receptor molecule. These data suggest that the beta subunit does not significantly influence the benzodiazepine, barbiturate, or steriod site pharmacologies of human GABAA receptor subtypes.