Paul J. Whiting

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.

Pharmacological characterization of a novel cell line expressing human α4β3δ GABAA receptors

The pharmacology of the stable cell line expressing human α4β3δ GABAA receptor was investigated using whole‐cell patch‐clamp techniques. α4β3δ receptors exhibited increased sensitivity to GABA when compared to α4β3γ2 receptors, with EC50s of 0.50 (0.46, 0.53) μM and 2.6 (2.5, 2.6) μM respectively. Additionally, the GABA partial agonists piperidine‐4‐sulphonate (P4S) and 4,5,6,7‐tetrahydroisothiazolo‐[5,4‐c]pyridin‐3‐ol (THIP) displayed markedly higher efficacy at α4β3δ receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. The δ subunit conferred slow desensitization to GABA, with rate constants of 4.8±0.5 s for α4β3δ and 2.5±0.2 s for α4β3γ2. However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. α4β3δ and α4β3γ2 demonstrated equal sensitivity to inhibition by the cation zinc (2–3 μM IC50). However, α4β3δ receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC50 for GABA antagonists SR‐95531 and picrotoxin, was similar for α4β3δ and α4β3γ2. Likewise, inhibition was observed on both subtypes at high and low pH. α4β3δ receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15‐4513 and bretazenil potentiated GABA responses on α4β3γ2 cells, and the inverse agonist DMCM showed allosteric inhibition of α4β3γ2 receptors. The efficacy of neurosteroids at α4β3δ receptors was greatly enhanced over that observed at α4β3γ2 receptors. The greatest effect was observed using THDOC with 524±71.6% potentiation at α4β3δ and 297.9±49.7% at α4β3γ2 receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at α4β3δ receptors versus α4β3γ2 receptors. We show that the α4β3δ receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics.

DISC1 and PDE4B Are Interacting Genetic Factors in Schizophrenia That Regulate cAMP Signaling

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3′,5′-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.

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.

Disrupted in Schizophrenia 1 and Nudel form a neurodevelopmentally regulated protein complex: implications for schizophrenia and other major neurological disorders

Disrupted In Schizophrenia 1 (DISC1) was identified as a potential susceptibility gene for schizophrenia due to its disruption by a balanced t(1;11) (q42;q14) translocation, which has been shown to cosegregate with major psychiatric disease in a large Scottish family. We have demonstrated that DISC1 exists in a neurodevelopmentally regulated protein complex with Nudel. The complex is abundant at E17 and in early postnatal life but is greatly reduced in the adult. Nudel has previously been shown to bind Lis1, a gene underlying lissencephaly in humans. Critically, we show that the predicted peptide product resulting from the Scottish translocation removes the interaction domain for Nudel. DISC1 interacts with Nudel through a leucine zipper domain and binds to a novel DISC1-interaction domain on Nudel, which is independent from the Lis1 binding site. We show that Nudel is able to act as a bridge between DISC1 and Lis1 to allow formation of a trimolecular complex. Nudel has been implicated to play a role in neuronal migration, together with the developmental variation in the abundance of the DISC1-Nudel complex, may implicate a defective DISC1-Nudel complex as a neurodevelopmental cause of schizophrenia.

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.

IDENTIFICATION OF A GABAB RECEPTOR SUBUNIT, GB2, REQUIRED FOR FUNCTIONAL GABAB RECEPTOR ACTIVITY

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.