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Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors*

Open AccessPublished:May 28, 2010DOI:https://doi.org/10.1074/jbc.M110.103309
      Nineteen GABAA receptor (GABAAR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABAARs is unknown. Here we report the discovery of a new structural class of GABAAR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABAAR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABAARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABAARs.

      Introduction

      γ-Aminobutyric acid (GABA),
      The abbreviations used are: GABA
      γ-aminobutyric acid
      GABAAR
      GABAA receptor
      FDD
      fragrant dioxane derivatives
      BZ
      benzodiazepine
      TMN
      tuberomamillary nucleus
      PN
      purkinje neurons
      VC
      Vertacetal®coeur
      VLPO
      ventrolateral preoptic area
      MAP2
      microtubule-associated protein 2
      WT
      wild type
      SCS
      salicylidene salicylhydrazide.
      the major inhibitory neurotransmitter in the brain, mediates inhibition via GABAA receptors (GABAAR), heteropentameric proteins constructed from subunits derived from several related gene families with six α-, three β-, three γ-, one δ-, one ϵ-, one π-, and one θ-subunit in mammals. In addition 3 rho (ρ)-subunits contribute to what have been called “GABAC receptors” (
      • Olsen R.W.
      • Sieghart W.
      ). According to the current model of the GABAAR structure the GABA-binding pocket is formed at the α/β-subunit interface, whereas the benzodiazepine (BZ)-binding pocket is located at the α/γ interface (
      • Sigel E.
      ) with the subunits arranged pseudo-symmetrically around the ion channel in the sequence γ-β-α-β-α anticlockwise when viewed from the synaptic cleft (
      • Baumann S.W.
      • Baur R.
      • Sigel E.
      ).
      Functional receptor compositions are restricted in their number and delineated on the basis of several criteria such as (i) capability of selected subunits to form a heteropentamer with defined pharmacological properties, (ii) a similar pharmacological fingerprint must be found in native receptors, and (iii) immunohistochemical co-localization of these subunits must be demonstrated at synaptic or extrasynaptic sites (
      • Olsen R.W.
      • Sieghart W.
      ). Only few subunit combinations are currently accepted as “identified” native GABAAR subtypes with β1-containing receptors not among them (
      • Olsen R.W.
      • Sieghart W.
      ) mainly because subunit-selective pharmacological tools are missing.
      In total, the GABAAR incorporates more than ten distinct modulatory binding sites targeted by anticonvulsive, antiepileptic, sedative, hypnotic, and anxiolytic compounds belonging to chemically different structural classes (
      • Bateson A.N.
      ,
      • Krasowski M.D.
      • Koltchine V.V.
      • Rick C.E.
      • Ye Q.
      • Finn S.E.
      • Harrison N.L.
      ,
      • Olsen R.W.
      • Chang C.S.
      • Li G.
      • Hanchar H.J.
      • Wallner M.
      ,
      • Rudolph U.
      • Möhler H.
      ) with some of them showing receptor type-specific actions. Benzodiazepine (BZ)-site agonists discriminate γ2-containing GABAARs from recombinant αβ-receptor types. Moreover, incorporation of different types of α-subunits into the receptor influences the sensitivity to different BZ site ligands (
      • Rudolph U.
      • Möhler H.
      ). Several modulators like propofol, pentobarbital, loreclezole, or etomidate are acting at the β-subunit of the GABAAR (
      • Rudolph U.
      • Möhler H.
      ,
      • Hill-Venning C.
      • Belelli D.
      • Peters J.A.
      • Lambert J.J.
      ,
      • Siegwart R.
      • Jurd R.
      • Rudolph U.
      ). The actions of propofol and pentobarbital are independent, the actions of loreclezole and etomidate are dependent on the type of β-subunit present in recombinant GABAARs: receptors containing β2- or β3-subunits are potentiated with an EC50 of about 1 μm while β1-subunit-containing receptors are potentiated with EC50 values above 10 μm (
      • Hill-Venning C.
      • Belelli D.
      • Peters J.A.
      • Lambert J.J.
      ,
      • Wingrove P.B.
      • Wafford K.A.
      • Bain C.
      • Whiting P.J.
      ).
      Searching for further modulators of GABAAR, we screened several libraries of odorants and report now the discovery of a new structural class of GABAAR modulators: fragrant (
      • Olsen R.W.
      • Sieghart W.
      ,
      • Baumann S.W.
      • Baur R.
      • Sigel E.
      )-dioxane derivatives (FDDs) that enhance the action of GABA with much higher potency at the β1-subunit-containing compared with the β2- or β3-subunit-containing GABAAR. With the help of FDDs we identify native β1-subunit-containing GABAA receptors in histaminergic neurons of the posterior hypothalamus that play a central role in the control of wakefulness.

      DISCUSSION

      We describe here a new class of positive GABAAR modulators with a unique specificity for receptors containing the β1-subunit. Following its detection in recombinant receptors, the β1-subunit selectivity of FDD is characterized in recordings from native neurons, providing first time evidence for the synaptic localization of β1-containing GABAARs in hypothalamic neurons and their role in somatic GABA responses. Pharmacological detection of native β1-containing GABAARs in hypothalamic neurons was supported further by immunohistochemistry and by an in vitro knockdown technique, demonstrating superior performance of FDD for the detection of β1-containing GABAARs in comparison to previously available pharmacological tools such as salicylidene salicylhydrazide (
      • Thompson S.A.
      • Wheat L.
      • Brown N.A.
      • Wingrove P.B.
      • Pillai G.V.
      • Whiting P.J.
      • Adkins C.
      • Woodward C.H.
      • Smith A.J.
      • Simpson P.B.
      • Collins I.
      • Wafford K.A.
      ). Recombinant receptors containing β1-subunits were nearly 6× more sensitive to FDD compared with receptors composed of β2- or β3-subunits. Macroscopic GABA-evoked currents recorded from Purkinje neurons lacking expression of the β1-subunit were 4.5 times less sensitive to FDD compared with the TMN neurons, while FDD potency in synaptic current modulation differed 7.1 times between these neurons. Although an ideal selectivity would be more than 10 times difference in potency, in all our different experimental approaches β1-containing GABAA receptors showed significantly higher modulation compared with the β1-lacking receptors on the whole concentration scale. The action of FDD was independent of γ-subunit and totally relied on the type of β-subunit present in the GABAAR. Two different α-subunits tested in recombinant GABAARs (α1 and α2) did not differ in their FDD sensitivity, however we cannot exclude that GABA-binding sites formed by other α-subunits may carry different properties. In keeping with the block of etomidate-evoked current by bicuculline in a study by Belelli et al. (
      • Belelli D.
      • Lambert J.J.
      • Peters J.A.
      • Wafford K.
      • Whiting P.J.
      ) and the block of pentobarbital-evoked current by bicuculline and gabazine (
      • Ueno S.
      • Bracamontes J.
      • Zorumski C.
      • Weiss D.S.
      • Steinbach J.H.
      ), FDD-evoked currents in our study were inhibited by gabazine, leaving open the possibility that the GABA-binding site is involved. However, an “allosteric model” suggests that gabazine inhibits the pentobarbital-current by reducing the probability of channel opening acting as an “inverse agonist” (
      • Ueno S.
      • Bracamontes J.
      • Zorumski C.
      • Weiss D.S.
      • Steinbach J.H.
      ).
      Decay kinetics of sIPSCs recorded from acutely isolated Purkinje and TMN neurons differed significantly in their modulation by FDD. While in most of the TMN neurons (86%) decay kinetics of sIPSCs were significantly prolonged by FDD 1 μm, and half-maximal prolongation was achieved at 14 μm, in Purkinje neurons minimal and half-effective concentrations were 20 and 100 μm, respectively. Thus, FDD reveal a synaptic localization of the β1-subunit in hypothalamic TMN neurons. As the β1-subunit was never expressed alone in TMN neurons (single cell RT-PCR data), it is likely, that β3- and β1-subunits are either co-assembled in the same receptors (
      • Sieghart W.
      • Sperk G.
      ) or present as separate populations carrying different functions. A recent study demonstrated reduced modulation by propofol (1.5 μm) of sIPSCs in TMN neurons recorded from β3N265M mice (
      • Jurd R.
      • Arras M.
      • Lambert S.
      • Drexler B.
      • Siegwart R.
      • Crestani F.
      • Zaugg M.
      • Vogt K.E.
      • Ledermann B.
      • Antkowiak B.
      • Rudolph U.
      ), supporting functional presence of β3-containing GABAARs (
      • Zecharia A.Y.
      • Nelson L.E.
      • Gent T.C.
      • Schumacher M.
      • Jurd R.
      • Rudolph U.
      • Brickley S.G.
      • Maze M.
      • Franks N.P.
      ); however, propofol effects on neuronal firing of TMN neurons were not investigated. Future studies employing mice with mutated GABAARs will answer the question about the relative contributions of all three β-subunits in controlling the firing of wake active hypothalamic neurons. Lesions in the posterior hypothalamus, which contains wake-on pacemaker neurons, are responsible for the encephalitis lethargica von Economo (
      • Von Economo C.
      ). GABA released from axons of ventrolateral preoptic area (VLPO) neurons during sleep (
      • Sherin J.E.
      • Elmquist J.K.
      • Torrealba F.
      • Saper C.B.
      ,
      • Sherin J.E.
      • Shiromani P.J.
      • McCarley R.W.
      • Saper C.B.
      ) inhibits two major groups of posterior hypothalamic wake-promoting neurons, the orexin- and histamine-producing neurons, which grow and can be recorded in posterior hypothalamic cultures (
      • Sergeeva O.A.
      • Andreeva N.
      • Garret M.
      • Scherer A.
      • Haas H.L.
      ).
      We demonstrate a high sensitivity of the firing of posterior hypothalamic neurons to FDD, only 3× lower than the propofol sensitivity. Moreover, incubation with β1-siRNA significantly decreased inhibition of neuronal firing by FDD at a large concentration range, but did not affect modulation by propofol. As a result, FDD became 11 times less potent in inhibiting neuronal firing compared with propofol after β1-siRNA treatment. Thus, the β1-containing GABAAR population controls not only synaptic integration but also the firing of hypothalamic neurons.
      We explored mechanisms of action of FDDs in recombinant GABAARs. The mode of GABAAR potentiation by FDDs resembles the action of propofol, targeting β-subunits directly. They modulate GABA responses and, at higher concentrations, gate GABAARs; the potentiation is markedly reduced at receptors carrying the β1M286W-mutation and the β3N265M mutation, whereas the β3N265S mutation shifts the sensitivity of the β3-subunit toward the β1-type. The possible interaction of FDDs with extrasynaptic GABAAR types, which mediate tonic inhibition and control neuronal firing (
      • Bonin R.P.
      • Orser B.A.
      ) awaits further investigation (
      • Meera P.
      • Olsen R.W.
      • Otis T.S.
      • Wallner M.
      ,
      • Bencsits E.
      • Ebert V.
      • Tretter V.
      • Sieghart W.
      ).
      What is the quantity of the β1-containing GABAAR population in the brain and what is the functional role of these receptors? According to an in situ hybridization distribution analysis of GABAARs (
      • Wisden W.
      • Laurie D.J.
      • Monyer H.
      • Seeburg P.H.
      ) the β1-subunit transcripts are found at moderate or low level in many brain areas (such as cortex, amygdala, septum, hypothalamus, striatum), where their expression level does not exceed expression of other β-subunits, except for the hippocampus, where β1-subunit transcripts are abundant, similar to the β3-subunit transcripts. Confusingly, despite high levels of extrasynaptic β1-containing receptors in the hippocampus, tonic inhibition (attributed to the extrasynaptic receptors) was found highly sensitive to the modulation by loreclezole (β2/3-preferring modulator) (
      • Mangan P.S.
      • Sun C.
      • Carpenter M.
      • Goodkin H.P.
      • Sieghart W.
      • Kapur J.
      ). Our single cell RT-PCR data, which are in agreement with in situ hybridization data on the expression of GABAARs in the hypothalamus (
      • Wisden W.
      • Laurie D.J.
      • Monyer H.
      • Seeburg P.H.
      ) showed an unexpected mismatch between transcription and function of β1-containing receptors, indicating that the role of these receptors in the brain is largely underestimated. Recently, β1/3-and ϵ-containing receptors with a unique pharmacological profile were described in noradrenergic neurons of locus coeruleus (
      • Belujon P.
      • Baufreton J.
      • Grandoso L.
      • Boué-Grabot E.
      • Batten T.F.
      • Ugedo L.
      • Garret M.
      • Taupignon A.I.
      ), however it was not clear, whether the β1- or the β3-subunit plays the dominant role. What is the consequence of the β1-subunit up-regulation found under pathological conditions such as hepatic encephalopathy, a liver disease accompanied by neurological symptoms due to increased GABAergic tone (
      • Li X.Q.
      • Dong L.
      • Liu Z.H.
      • Luo J.Y.
      )? The novel class of GABAAR modulators described here will help to answer this question.
      During systemic propofol administration cFos expression in TMN neurons decreases, a GABAAR antagonist injected into TMN can antagonize anesthesia and local injection of propofol into TMN causes sedation. The silencing of wake-promoting neurons in the hypothalamus has been connected with the sedative component of anesthesia (
      • Nelson L.E.
      • Guo T.Z.
      • Lu J.
      • Saper C.B.
      • Franks N.P.
      • Maze M.
      ). Thus the hypothalamus is a center for sleep-waking regulation that integrates immobilization by anesthetics. The role of the β1-subunit in anesthesia remains to be elucidated.
      In conclusion, our findings provide a new pharmacological tool for the phenotype of β1-subunit-containing GABAARs. After validation of subunit selectivity of FDD in recombinant and native GABAARs, we determined the hypothalamus as an important target for GABAAR modulators interacting with the β1-subunit.

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