Ruth M. McKernan

Which GABAA-receptor subtypes really occur in the brain?

GABAA receptors are a heterogeneous family of ligand-gated ion channels responsible for mediating inhibitory neurotransmission in the CNS. Since the identification of mammalian cDNAs encoding 13 GABAA-receptor subunits, the composition of native receptor molecules and their localization in the brain has been an area of intense study. We conclude that the number of major subtypes is probably less than ten but their physiological roles have yet to be clearly defined and this represents the next step in GABAA-receptor research.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABAA receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (α1 H101R) with a diazepam-insensitive α1 subtype and a selective BZ site ligand, L-838,417, to explore GABAA receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the α1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABAA receptor subtypes in distinct neuronal circuits.

NMDA receptor pathways as drug targets

Since the mid 1980s, there has been a great deal of enthusiasm within both academia and industry about the therapeutic potential of drugs targeting the NMDA subtype of glutamate receptors. That early promise is just beginning to translate into approvable drugs. Here we review the reasons for this slow progress and critically assess the future prospects for drugs that act on NMDA receptor pathways, including potential treatments for some major disorders such as stroke and Alzheimers disease, for which effective therapies are still lacking.

Molecular and Functional Diversity of the Expanding GABA-A Receptor Gene Family

ABSTRACT: Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter γ‐aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA‐A receptor subtypes exists, generated through the coassembly of polypeptides selected from α1‐α6, β1‐β3, γ1‐γ3, δ, ɛ, and π to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA‐A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA‐A receptor gene family, which we have termed θ, which appears to be most closely related to the β subunits. The structure, function, and distribution of θ‐containing receptors, and receptors containing the recently reported ɛ subunit, are described.

Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.

Structure and Pharmacology of Vertebrate GABAA Receptor Subtypes

Publisher Summary This chapter reviews GABAA receptors and describes how the techniques of molecular neurobiology enabled a revolution in the understanding of the GABAA receptor. In common with the other members of the ligand-gated ion channel family, the function of the GABAA receptor is modulated by phosphorylation. Indeed, purified native GBAA receptor protein can be phosphorylated in vitro by cyclic AMP-dependent protein kinase A (PKA) and calcium/phospholipiddependent protein kinase C (PKC). Analysis of the deduced amino acid sequences of GABAA receptor subunits indicates that the large cytoplasmic loop domains contain consensus sites for phosphorylation by PKA (human α3,α 4, α6, β1, β2, β3,γl , γ2, and γ3), PKC (all human subunits), and tyrosine kinase (γl, γ2, γ3) . It is this domain of the nAChR that undergoes phosphorylation. The pentameric structure of the GABAA receptor is based on analogy with the nicotinic receptor, the physicochemical properties of the solubilized receptor, and electron microscopic studies of purified receptor preparations. A diverse range of both naturally occurring and synthetic compounds can allosterically regulate GABAA receptors. By using recombinant receptors, it is possible to study the roles played by individual subunits in the actions of many of these compounds and indeed, in some cases, individual amino acids located at the binding sites have been identified.

Evidence for a Significant Role of α3-Containing GABAA Receptors in Mediating the Anxiolytic Effects of Benzodiazepines

The GABAA receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that α2-rather than α3-containing GABAA receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an α3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2′-difluoro-5′-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an α3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders α2-containing receptors BZ insensitive (α2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of α3-containing GABAA receptors is sufficient to produce the anxiolytic effects of BZs and that α2 potentiation may not be necessary.

α4β3δ GABAAReceptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential

Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

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.