Sally-Anne Thompson

Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination.

1 Human GABAA receptors containing different α and β subunits with a γ2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two‐electrode voltage‐clamp method. 2 Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype. 3 Potentiation of GABA by pentobarbitone occurred over the same concentration‐range for all the subunits with affinities in the range of 20–35 μm. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on α1β2γ2s to 536% on α6β2γ2s. 4 Examination of the direct effect of pentobarbitone revealed that the type of α subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an α6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other α subunits ranged between 45% of maximum GABA for α5β2γ2s to 82% for α2β2γ2s. The affinity of the direct action of pentobarbitone on α6β2γ2s was 58 μm compared to affinities for the other α subunits ranging from 139 μm on α2β2γ2s to 528 μm on α5β2γ2s. 5 The type of β subunit present did not influence the direct action of pentobarbitone to the same extent as the α subunit. There were no significant differences between affinity or efficacy on oocytes expressing α6 and γ2s with β1, β2 or β3. Affinities and efficacies on oocytes expressing α1 and γ2s with β1, β2 or β3 were significantly different with pentobarbitone having a higher affinity and efficacy on α1β3γ2s followed by α1β2γ2s and then α1β1γ2s. 6 The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site. 7 For the first time the influence of the various α and β subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing α6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.

Mutation at the putative GABAA ion‐channel gate reveals changes in allosteric modulation

We have mutated a conserved leucine in the putative membrane‐spanning domain to serine in human GABAA β2 and investigated the actions of a number of GABAA agonists, antagonists and modulators on human α1β2ΔL259Sγ2s compared to wild type α1β2γ2s GABAA receptors, expressed in Xenopus oocytes. The mutation resulted in smaller maximum currents to γ‐aminobutyric acid (GABA) compared to α1β2γ2s 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 α1β2ΔL259Sγ2s 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 α1β2ΔL259Sγ2s 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.

Mechanism of action of general anaesthetics--new information from molecular pharmacology.

Major progress in our understanding of the mechanisms of anaesthesia has been made during the past year. Several key advances in defining very specific sites of action on ligand-gated ion channels have been described. Furthermore, new techniques have become available for addressing the identification of binding sites and transduction mechanisms on these receptors. The discovery that anaesthetics affect a recently identified family of potassium channels could also lead to major new findings in the next few years.

Salicylidene salicylhydrazide, a selective inhibitor of β1-containing GABAA receptors

A high‐throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of α2β1γ1θ GABAA receptors with a maximum inhibition of 56±5% and an IC50 of 32 (23, 45) nM. Evaluation of this compound using patch‐clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the β1 subunit (e.g. maximum inhibition of 68.1±2.7% and IC50 value of 5.3 (4.4, 6.5) nM on α2β1γ1 receptors). The presence of a β1 subunit was paramount for the inhibition with changes between α1 and α2, γ1 and γ2, and the presence of a θ subunit having little effect. On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at α1β1γ1θ 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 EC50 or Hill coefficient. In addition, SCS inhibited pentobarbitone‐induced currents. Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. 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. In conclusion, SCS is unique in selectively inhibiting GABAA receptors containing the β1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the β1 subunit and the lack of interaction with a range of GABAA receptor modulators suggests that SCS is interacting at a previously unidentified site.

Evidence for an enhancement of excitatory transmission in adult CNS by Wnt signaling pathway modulation.

The role for Wnt signaling modulation during synaptogenesis, neurogenesis and cell fate specification have been well characterized. In contrast, the roles for Wnt signaling pathways in the regulation of synaptic plasticity and adult physiology are only starting to be elucidated. Here, we have identified a novel series of Wnt pathway small molecule modulators, and report that these and other small molecules targeting the Wnt pathway acutely enhance excitatory transmission in adult hippocampal preparations. Our findings are consistent with a pre- and postsynaptic site of action, leading to both increased spontaneous and evoked neurotransmission that occurs in a transcription-independent fashion.

Functional characteristics of recombinant human GABAA receptors containing the ϵ-subunit

Abstract (1) This paper further examines the functional characteristics of recombinant human GABA A receptors containing the ϵ -subunit expressed in Xenopus oocytes. (2) α 1 β 1 ϵ receptors are not modulated by benzodiazepine ligands or by a number of hypothalamic hormones. (3) The intravenous anaesthetic agents pentobarbital, propofol and etomidate all potentiate sub-maximal GABA currents (EC 20 ) to a similar degree in α 1 β 1 ϵ and α 1 β 1 γ 2s receptors. (4) Direct activation by pentobarbital produced a similar maximum response on α 1 β 1 ϵ and α 1 β 1 γ 2s, however, both the EC 50 and slope were lower on α 1 β 1 ϵ compared to α 1 β 1 γ 2s. (5) These results describe a novel pharmacology for recombinant α 1 β 1 ϵ receptors.

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.