European Journal of Pharmacolo~" ~ Molecular PP,armaeologI" Section. 189 (1990) 337-340

337

Elsevier EJPMOL 80037 Short

communication

Identification of the ~,2-subunit protein in native GABAAreceptors in brain D i e t m a r B e n k e ~, S t e p h a n M e r t e n s ~, A r n o l d Trezciak -, ' D t"e t e r G i t l e s s e n z a n d Hant~s M o h l e r t 1 Institute t~f Pharmacology; Unicersity of Zurich. Zurich. Switzerland- aad -" Central Research Deparrmem. t'~ ttoffr~ana-La Roche, Basel. Switzerland

Received2 July 1990, accepted 31 July 1990

GABAA receptors with functional benzodiazepine receptors have been reconstituled by coe×pression of a-. r- and -/-subunit eDNAs. In brain, proteins of the a- and/Lsubunits, but not the -t2-subunit have been identified. Using an antipeptide antiserum, we now demonstrate that the "r2-subunit is a protein of 43 kDa. it is pre~nt in at least 50% of GABAA receptors, as shown by immunoprecipitation. GABA A receptor: 72-Subunit; Amipeptide antibodies

l. Introduction

GABA a receptors are GABA-gated C I - channels which serve as central control elements for the pharmacological regulation of anxiety, vigilance, muscle tension and epileptiform activity effected mainly via the benzodiazepine receptor (BZR). The BZR is unique in that it allows the modulation of G A B A A receptor function in opposite directions induced by agonists or inverse agonists, respectively. While initial biochemical evidence suggested that G A B A A receptors are composed of a- and B-subunits, eDNA cloning revealed an unexpected heterogeneity of suhunits comprising at least six a-subunits, three B-subunits, one "/- and one 8-subunit (Schofield et al.. 1990; Mohler et al., 1990). Functional expression of ul- and /31-subunits resulted in GABA-gated channels which did not respond to ligands of BZR or showed a functionally restricted response to these ligands (Malherbe et al., 1990a, in press).

Correspondence to: Harms Mohler, Ph.D., Institute of Pharmacc?ogy.Universityof Zurich, Glofiastr. 32, CH-8006Zurich.

However, when the 72-subunit was co-expressed together with the a l - and/~t-subunit, the resulting receptors displayed a fully functional BZR response: the GABA-induced current was enhanced by the agonist diazepam and reduced by the inverse agonist methyl-4-ethyI-6,7-dimethoxy-/~carboline-3-carboxylate (DMCM) (Pritchett et aL, 1989; Matherbe et at., 199"3b. in press). However, previous biochemical studies had not provided any evidence for the existence of a -t2-subunit protein in native GABAa-receptors. We now demonstrate the presence of the "f2-subanit protein in GABAa-receptors purified from rat and bovine brain.

2. Materials and methods 2.1. P r e p a r a t i o n o f antibodies

The "12-snbuni~ peptide (see Results) was synthesized by the solid phase method and coupled to keyhole limpet hemocyanine (KLH) (Wang, 1973L The corresponding 72N-antiserum was raised in rabbits ( K A O H strain, BRL).

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338 Bovine

2.2. Gel electrophoresis and Western blot analysis

1

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blmting was performed as described elsewhere (Benk ,~ et al., 1989). GABA A receptor preparations were purified from bovine cerebral cortex or rat whole brain according to Sigel et al. (1983) and Schoch and Molder (1983), respectively. For immunostaining, the 72N-antiserum was diluted 1 : 500.

2.3. Immunoprecipitation Purified GABA A receptor preparations were incubated overnight at 4 ° C with various amounts of 72N-antiserum (1-15 #l) in a total volume of 120 /tl. After addition of 50 /tl Pansorbin (Calbioehem) followed by incubation for 1 h at room temperature and centrifugation, radioligand binding assays ([3H]flumazenil and [3H]muscimol) were performed to test for the presence of G A B A g receptors in the resuspended precipitate as described by Schoch et al. (1985).

3. Results The availability of the 72-subunit e D N A sequence of rat brain (Shivers et al., 1989; Malherbe et al., 1990b, in press) provided the opportunity to raise an antiserum against a 72-subunit polypeptide. A peptide corresponding to the N-terminal sequence ( p G l u - K S D D D Y E D Y A S N K T ) of the 72-subunit was synthesized with an additional cysteine residue added at the C terminus for coupling the purified peptide (72N-peptide) to KLH. This peptide is selective for the y2-subunit sequence as compared to other known subunits of the GABAA-, glycine- and nicotinic acetylcholine receptor. The corresponding 72N-antiserum, raised in rabbits, showed a high titer as tested by ELISA using the peptide or purified G A B A A receptor preparations as antigens. The y2-subunit protein was identified in GABA a receptor preparations which were purified fram rat or bovine brain by a benzodiazepine-affmity chromatography coMran (Sigel et al., 1983; Schoch and M~hler, 1983). Since most

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Fig. 1. Identification of the 72-subunit in purified GABAA receptor preparations by Western blotting. GABAa receptor preparations purified from whole bovine brain (lanes 1-3) and rat brain (lanes 4-5) were subjected to SDS-PAGE (0.4 pmol [3Hlflumazenil binding sites per lane). Lane 1: Coomassie staining; Lanes 2-5: immunostaining with y2N-antiserum in the absence(lanes 2, 4) and pre~ence(lanes 3, 5) of 72N-peptide (25 ~Lg/ral). G A B A A receptors are thought to contain benzodiazepine binding sites, it can be assumed that these preparations comprise nearly all G A B A a receptor subtypes and are suitable to test for the presence of the 72-subnnit. When G A B A A receptor preparations purified from rat brain were subjected to SDS-PAGE followed by Western-blotting and immunostaining with the 72N-antiserum, a single protein band of 43 k D a was stained (fig. 1, lane 4). The immunoreaction was specific since it was inhibited in a concentration-dependent manner by the 72N-peptide (fig. 1, lane 5). A protein band of the same size was labeled w h ~ crude membrane preparations of rat brain were used for Western blotting. Likewise, when G A B A A receptors purified from bovine brain were used for immunoblotting, a 43-kDa band was stained (fig. 1, lane 2) and the immunoreaction was inhibited in the presence of the "t2N-peptide (fig. 1, lane 3). Thus, the 72-subunit immunoreactivity is present in a protein of 43 k D a in both bovine and rat brain G A B A A receptor preparations. In some, but not all, Western blot experiments, weak immunostaining was apparent at a band of 67 kDa. However, this staining was nonspecific since it was not displaced by the 72N-peptide (fig. 1, lane 5). A weak displaceable staining, observed in some pre-

339 parations at about 90 kDa and 200 kDa, is presumably due to receptor aggregation. The presence of a 43-kDa protein band in purified receptor preparations can also be demonstrated by Coomassie staining (fig, 1, lane 1). The immunoreactive protein is a frequent receptor constituent as shown by immunoprecipitation experiments. Using radioligand binding to monitor GABA A receptors purified from rat brain, it was found that an increasing number of binding sites were precipitated in the presence of ir~creasing amounts of the ~,2-N-antiserum, with a plateau being reached at 49 + 4% of [3H]flumazenil binding sites and 50 + 6% of [3H]muscimol binding sites, Thus, at least half of the GABA A receptors contain the -/2-subunit immunoreactivity.

calculated M~ of the ungtycosylated ~2-subunit polypeptide (48 kDa) and its size on SDS-PAGE (43 kDa) is not unusual and has also been observed with other proteins, e.g, the a-subunit of the nicotinic acetytchotine receptor with its calculated polypeptide mass (50 kDa) being larger than the size of the native subunit on SDS-PAGE (40 kDa; reviewed by Hucho, 1986). A band of 43 kDa is apparent in GABA a receptor preparations stained with Coomassie blue (fig. 1. lane t), atthough the band is less intensely stained than the 50-kDa band. The identification of the ),2-subunit will open new opportunities to identify by crosslinking studies its neighboring subunits and thereby help to clarify the subunit structure and stoichiometty of GABAa receptors in brain,

4. Discussion References An antiserum raised against a selective "t'2-subunit peptide immunoreacted with a protein of 43 kDa present in GABA A receptor preparations of rat and bovine brain, The immunoreaction in Western blots aad immunoprecipitation was specific since it was inhibited by the peptide antigen. These results suggest that the 43-kDa protein represents the y2-subunit protein. A possible crossreactivity of the antiserum with yet unkno~n subunits with higla homology to the N-terminal sequence of the ~,2-subunit, however, cannot be excluded. The identification of the y2-subunit as a 43-kDa protein in native GABA A reccptors in rat and bovine brain is of major relevance since, in reconstituted receptors, the ~,2-subunit in combination with a- and fl-subunits confers full sensitivity to ligands of ;h~ BZR (Pritchett et al., 1989; Malherbe et al., 1990b, in press). Although a- and/Lsubunit proteins (50 and 55 kDa) were apparent in purified receptors n various biochemical studies, there was no evidence for a major band accounting for the "¢-subunit. This was initially thought to be due to the possible co-migration of the 7-subunit v.,ith the a- or fl-subunits. Our results suggest that the y2-subuni! is a protein of 43 kDa (fig. t, lanes 2, 4) distinguishing it in size from the ~- and r-subunits known so far. The discrepancy between the

Benke. D.. J~ Stahmer and H. Breer. 1989, Mon~!on~ antibodies detecting regulator" polypeptidesof ~he in~t neuronal acetylcholine receptor. J. Exp. Biol. 147, 329. Hucbo. F, 1986, The nicotinic needy]choline receptor and its ion channel, EuroF,-eanJ. Biochem. 158. 2tl. Malhefne. P., A. Draguhn.G. Mukhaup, K. Beyreatherae,d H. Mohler. 1990m GABA• recep!orexpr~sed from rat bra/n a- and fl-subunitcDNAs displayspotentiation by benzt~iiazepine receptor ligands. MoL Brain Res., in press, Malherbe. P., E, Siget, R. Bunt, E. PerssohmJ.G. Richards and H. Mohter. 19eA3b.Functional characteristics and sites of gone expression of the ~!-, fit-, -/2-isofnrmof the rat GABA.~ receptor, L Neurosci., in press, Mohter, H., P. Malherbe. A. Dragubnand J.G, Riehards, 1990, GABAa receptors: structural requirementsmad st,toof gone expression in mamw~t~anbrain. Neurochem. Re~ 15, 199. Pritchen, D,B., H. Sontheimer. B.D. Shivers, S, Yraer, H, Kettenmann, P.R. S,chofield and P.H. Seeburg t989. Importance of a novel GABAA receptorsubuni~,for benz(xliazepine pharmacology.Na~.ure338, 582. Schoch, P. and H. Mohler. I983. Purified benzodiazepine receptor retains modulation by GABA, European J. Pharmacol. 95. 323. Schoch. P.. J.G. Richards, P. H~ifing, B. Takacs. C, St~ihlLT. Staebelin. W. Haefely and H. Mob!or.I985, Co-localization of GABA~,-receptor and benzodiaz*-pine receptor in the brain shownby monoclona!antibodies. Nature 314, t68, Schofield. P.R., B.D. Shiversand P.H. Seeburg. 1990. The role of receptor subtypediversity in the CNS TrendsNe~roseL I3,8. Shivers. B,D., t. Kdlisch. R. SprengeL H. Somheimer, M. K/3hler, P.R. Schofield and P.H. Seeburg. t%9, T~o novel

340 GABA n receptor subunits exist in distinct neuronal subpopulations, Neuron 3+ 327. Sigel, E.. F.A. Stephenson+ C. Mamalaki and E.A. Barnard+ 1983, A y-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex: purification and partial characterization+ J, Biol, Chem. 258. 6965.

Wang. S.-S., 1973, p-Alcoxybenzyl alcohol resin and p-AIcoxybenzyl-oxy carbonylhydrozide resin for solid phase +ynthesis of protected peptide fragments. J. Am. Chem. Coc. 95, 1328.

Identification of the gamma 2-subunit protein in native GABAA receptors in brain.

GABAA receptors with functional benzodiazepine receptors have been reconstituted by coexpression of alpha-, beta- and gamma-subunit cDNAs. In brain, p...
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