Identification and immunohistochemical mapping of GABAA receptor subtypes containing the δ-subunit in rat brain

Volume 28~, number I. 14~i49

F~BS (~97,tt~

May t991

~, 1991 Fctler,'ltlon t~t European Biod~emlc=ql Societies; 11Ol4~'~M~1~$),$t~ A IJONL~ 0014~'19~910044"/F

Identification:and immunohistochemical mapping of GABAA receptor subtypes containing the 6-subunit in rat brain D. Bcnke =, S. Mertens~ =.A. Trzeciak =. D, Gillesset~= a n d H, M o h l e r ~ t Institute o f Ph¢trma¢olagy, University o fZiirtch, CH.SOO6 Z~Trlcl~. Swlt-.erland and :Phm'm¢~ Research Department. F. [foJJhtann. L+t Roche. CIt.400~ B¢~Ie; Swit:¢rhmd Received 25 March 1991 S)'naptic inhibition in brain is mainly mediated via GABA^ rc¢¢ptont which di~pl:ty a strikin~ strtt¢tural betero~tenCity. A novel type of GABA^ receptor subtmit. ~he 6.subtmit. has recently been described b=~d on molecular donin~l of'lt~ eDNA Ill. To identify the pre ealenee and distribution of GABA^ receptors which contain the 6.subunit protein in sltu polyclomd site.directed antiscm were developed altainsl thre~ synthetic pcptides

derived rorm the rat 6-subtmit eDNA.:~equcnec, All anti~ra ~pccifically recognized a ~4 kDa protein i~ GABA^ receptor prepanttion~, Nearly 30~ of the GA BA,,, reccptor~ contained tl~e6.suhunit irnmunoreaetivity and displayed high affinity GABA and birth atllnity bcnzodiazepine bindinlt sites as ~hown by immunopt¢cipitation, Receptors which contain ~he di-subunit were irnmunohistochemically shown to be restricted to a few brain areas such as: the cerebellum, thalamus and dentate gyrus ol" the hippocampal formation, Thus. those neurons which express GABA^ receptors with a 6.sUbUnit have now been vimtalized arid made accessible for a functional analysis of ttlis GABA^ receptor subtype in situ, GABA^ receptor; ~$-Subunit; Ant lpcptide antisera; Western btottlnlg lmmunc~precipitation; lmmunohistochemistry

1. INTRODUCTION Inhibitory neurotransmission in mammalian brain is mainly mediated via GABA^ receptors. They constitute GABA-gated hetero-oligomeric receptor-channel complexes wlfich can be allosterically modulated by various clinically important neuroaetive drugs, notably by ligands of the benzodiazepine receptor and by barbiturates [2]. Despite their physiological and pharmacological relevance, the structure of GABA^ receptors is yet unresolved. Initial biochemical and immunochenaical characterization of the GABA ~, receptor pointed to a 250 kDa 'eceptor complex consisting of aand ~3-subunits (reviewed in [3]), Subsequent molecular cloning of the c~. and ,3-subunit cDNAs led to the discovery of not only multiple a- and B.subunit isoforms ( a t - 6 , /3~,31 but also of multiple 3,-subunits (3't- 2) and a +5.subunit (reviewed in [4-6]). To analyze the subunit composition of various GABAA receptor subtypes in situ, subunit-specific antibodies are being used. The a~-, c~2-, c~3-, u6-, "YZ-and ~32/.~-subunits were identified as proteins of 50, 53, 60, 57, 43 and 56/57 kDa, respectively [7-18]. A frequent association of the c~-, 3z/~- and 3,z-subunits was recently demonstrated for GABAA receptors in situ [13] suggesting that this subunit combination is of physiological relevance. Coexpression o f this subunit combination (ect, /3z, 3,2) Correspondence address: H, Mohler, Institute of Pharmacology, University of Ztirich, Glofiastr, 32, CH.8006 Ztirich. Switzerland~ Fa~: (411 (I) 261 5686.

Published by Elsevier Science Publishers B.V.

resulted in GABA-gated chloride channels which were modulated by benzodiazepine receptor tigands [19,20]. In contrast to the a-,/~- and 3,-subuni~s, no information exists on the size, distribution and functional role of the 8-subunit protein in situ. In recombinant receptors, this subunit, which was identi fled by molecular cloning[ 1], formed GABA-sensitive homo-oligomeric ion channels but failed to convey benzodiazepine receptor modula.~ tion wheri co-expressed with o~- and B-subunits [1], However, it remained unclear to what extent the <3. subunit is a constituent of GABAA receptors in rico, possibly denoting receptor subtypes which lack certain modulatory responses to drugs. To approach this question we developed antisera against three different peptide sequences unique to the 8-subunit and report here on the identification, size and distribution o f the ~5subunit protein as integral component in a subset of GABAA receptors in vivo. 2. MATERIALS AND METHODS Three subunit-specif[c peptides derived from the rat ~5-subunit eDNA sequence [I] were synthesized: peptide 6(1-17)(sequence pyroglutamyl-PH HGARAMNDIGNYVG), peptide 6(179-189) (sequence YWSENQEQ1HG) and peptide //(316-322) (sequence HFNADYR) [21,22]. An additional cysteine was added to the Cterminus (peptides 6(1-17) and 6(179-189)) or N.terminus (peptide 6(316-322)), For antibody production the peptides were coupled via the additional cy~teine tokeyhole timpet haemocyanine (KLH) [23] and about 100 og conjugate emulsified 1:2 in Freund's complete adjuvant were used for immunization of rabbits (KOHA strain, BRL) as described previously [12-131. The development of an immune response was monitored by enzyme-linked immunosorbent assay

145

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tisera were raised against three different cDN&dcrivcd peptide sequences unique to the rat &subunit. The Nterminal. pcptide (residues I-17), as well as a pcptide from the proposed extracellular domain (residues 179-189) and a pepcide from the proposed intracellular loop located between the transmembrane spanning regions M3 and M4 (residues 316-322) were synthcsizcd and coupled to KLH and used as antigens, High titer antisera were already obtained two weeks after the first booster injection as tested in ELISA using the respcctive peptidcs as antigens. To identify the size of the &subunit protein GABA,, receptors purified from whole rat brain were tested with the antisera on Western blots. All three antisera selcctively recognized a 54 kDa polypeptidc (Fig. I, lanes 1,3,5). Co-incubation of the antisera with the rcspcctivc peptides blocked the immune reaction in all three cases documenting the specificity of the antibody reactions (Fig. 1, lanes 2,4,6). The apparent size of the native protein (54 kDa) exceeds that of the deduced amino acid sequence of the &subunit cDNA (49 kDa) [I] suggesting that a carbohydrate moiety may be attached at one or both potential N-linked glycosylation sites withirt the Ssubunit sequence contributing to the apparent molecular size of the native protein, Two of the antisera, the &l-17)- and the 6(179-189)antiserum, recognized the d-subunit in the native conformation as demonstrated by immunoprecipitation cxperiments. As illustrated for the b(l-17)~antiserum, incubation of purified GABAA receptor preparations with increasing concentrations of the antiserum resulted in saturable immunoprecipirarion of GABA+, receptors ag monitored by [3H]flumazenil and [‘Hlmuscimoi radioligand binding (Fig. 2A). A maximum of 28 3-7% 01 = 7) of I’H]flumazenil binding and 26 -+.6% (n = 5) of [3H]muscimol binding was precipitated with the 6( l-1 “I)-antiserum. Similar results were obtained with the 6(179-189)-antiserum which yielded a maximum precipitation of 30 I 2% (n = 4) of [3H]flumazenil bin146

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3. RESULTS AND DISCUSSICN

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ding and 21 f 6ofo(n = 3) of [lH]muscimol binding. The specificity of the immune reaction was 9sscssed in peptide competition experiments. When increasing concentrations of the pcptidc 6(1-17). were added to the a( I-17)-antiserum the immunoprccipitation of GAB& receptors was prcvcntcd in a dose-dependent manncc (Fig. 2B). Thus, a subpopulation of nearly 30% of GABA,, receptors appears to contain the J-subunit, To determine the regional distribution of the Ssubunit protein, crude membrane fractions from difa

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Volume 21~;t, number I

FEI~S LETTERS

ferent brm=tin areas were analyzed for immunoreactivity in Western blo|s, In membrane~ or' all brain regions e x . amined, a sinille protein band o f 54 kDa was labeled with zinc~(|-lT).anttserum, confirmin~ the results obtained with the purified G A B A ^ receptors (Fil3, 3), Although the ~.subunit immunoreactivily was present in all brain regions examined the intensities of signals varied. Strongest labdint~ was observed in the cerebellum followed by thalamuslhypothalamus, olfactory bulb and cerebral cortex, whereas weaker signals were detected in the striatum, hippocampal formation and brainstem. To quantify the amount of G A B A ^ receptors that contain the ~-subunit in the various brair~ re,ions immuno. precipitation experiments were performed using crude deoxycholate extracts of brain membranes (Table 1). The amount o f GABA^ receptor precipitated from extracts of whole brain (21 ± 2o10 [~Hlflumazenil binding, n = 3 ) w a s slightly lower than that precipitated from purified GABA^ receptor preparations (nearly 30070). In line with the regional prevalence of the 8-subunit detected in Western blots, highest immunoprecipitation was observed in extracts of the olfactory bulb, cerebellum and thalamus/hypothatamus, whereas in the cerebral cortex, striatum and hippocampal formation significantly less receptor was precipitated (Table l). These results suggest that the 5-subunit protein may be differentially expressed in various brain regions. To identify those neuronal populations that express GABA^ receptors containing the ~5-subunit, tmmunohistochemical experiments were performed +in parasagittal cryostat sections of rat brain. The ~-subunit im. munoreactivity was most abundant in the granular layer of the cerebellum with mainly somatic staining in a subset of cells (Fig. 4D), followed by thalamus, dentate gyrus of the hippocampal formation and olfactory tubercle (Fig. 4A) In other brain regions, e.g. olfactory bulb, caudate putamen, ventral pallidum and cerebral cortex, only weak staining was detected (Fig. 4A).:The specificity of immunostaining was assessed in peptide competition experiments. When the peptide 5(1-17) was added to the antiserum prior to the incubation procedure no immunostaining was observed (Fig. 4B). In general, the intensity of the ~-subunit signal observed immunohistochemically in various brain regions was in congruence with that obtained by Western blotting or immunoprecipitation. Furthermore, the immunostaining pattern of the ~5-subunit protein (Fig. 4A) correlated well with the expression pattern of the 5-subunit mRNA [1]. For instance, in the hippocampal formation, the immunoreactivity of the dendritic area of the granule cells of the dentate gyrus (Fig. 4A,C) is correlated with the in situ hybridization signals in the corresponding cell body layer [1]. Likewise, in the cerebellum, strong immunoreactivity was displayed by the granular layer (Fig. 4A) which also showed very high in situ hybridization histochemistry signals for the ~5-subtmit mRNA [1].

May 1991

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~everntbrain area~ wereapplied to eael~lan~and tact+batedwtlh the /~179-189).antiserum (dlluted I: 1000)+ In the molecular layer no signals could be detected either immunohistocl~emically (Fig. 4D) or by in situ I~ybridization histochemistry [1]. The ~5-subunit protein is most prominently expressed in the cerebellar granular layer, an area which is characterizedby high levels of high affinity muscimol binding and low levels o f benzodiazepine receptor binding [26]. It had therefore been suggested that the cSsubunit may constitute a GABAA receptor subtype that lacks high affinity benzodiazepine binding sites [1]. This was supported by the observation that the ~subunit, :when co-expressed with ~- and/3-subunits, was not able to confer high affinity benzodiazepine binding to recombinant GABA~ receptors [11. However, our immunoprecipitation data clearly show that GABAA receptors which contain the ~5-subunit immunoreactivity display bot!~ high affinity GABA sites and high affinity benzodiazepine binding sites in all brain areas tested. Since functional benzodiazepine receptors are generated it~ recombinant receptors by co-expression o f a-, ~3-and ./z-subunits, it is conceivable that the 8subunit may be associated with a-,/3- and 7z-subunits in Table I lmmunoprecipitation of GABAA receptors containing the ~-subunit from crude deoxycholate extracts o f various regions of rat brain Brain Region Olfactory bulb Cerebellum Thalamus/hypothalamus Cerebral cortex Striatum Hippocampus

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Deoxycholate extracts from membranes of various brain areas (200 jd) were incubated with an amount of ~(179-189)-antiserurri(t0 #l) which leads to ma×imumimmunoprecipitation.The results are expressed as percentageof specific['~H]flumazenilbinding; 100%values correspond to the sum of the radio|igand binding in the immunoprecipitate and in the corresponding supernatant. Nonspeeific immunOprecipitationwas determinedby replacingthe antiserum by preimmuneserum or buffer. The valuesare means _ SD from three experiments analysedin tri plicateS. 147

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Fig. 4. I mmunohistochemical localization of- 8-subunit immunoreactivity in parasagittal rat brain sections incubated witl~ affinity-purified ~(1- t 7)antiserum (diluted 1:10). (A,B) Macroscopic view of staining in the absence (At a n d presence (B) of 60 tag/rnl peptide 8(1-17). (C) lmmunostaining in the thalamus and the hippocampal formation. (D) Microscopic view of cerebellar stainhtg. Note the intense staining in cerebellar granule cells and the lack of staining in the Pt, rkinje cell layer and the molecular layer. Positive signals tn (A,B,C) correspond to the wlfite areas, in (D) to tire black areas. Bars: (A,B) 2,0 ram; (C) 0.8 mm; (D) 20 tma. Abbreviations: Cx, cerebral cortex: 13, dentate gyrus; H, hippocampus; G, granular layer: M, molecular layer; Ob, olfactory bulb; P, Purkinje cells; T, thalamus.

vivo. This view is supported by the strong immunoreactivity in the cerebellar granular layer observed not only f o r the 8-subunit but also for the c~~,, B2/3 and -/2-subunit proteins [13,15]. In summary, we have demonstrated that the 8subunit is an integral protein o f 54- kDa in a subset o f native GABAA receptors comprising up t o 30% o f GABAA receptors in different brain areas. Neuronal populations which express the 8-subunit were identified immunohistochemically in situ ancl are now amenable to a functional analysis of this GABAA receptor subt48

type. Mapping the various subunit combinations is part o f the attempt to establish a s t r u c t u r e / f u n c t i o n relationship for the various GABAA receptor subtypes in the brain. REFERENCES [11 Shivers; B.D,, Killisch, I., SrJrengel, R.. Sontheimer, H., Kohlcr, M., Schofield, P.R, and Seeburg, P.H. (1989) Neuron 3. 327-337, [21 Olsen, R,W, and Venter, J.C. (1986) Benzodiazepine-GABA Receptors and Chloride Clmnnels: Structural and Functional Properties, Alan R. Liss, New York.

IF;“ERS RETTEfES

Velum4 2&U,number I 131 Xtsphawwt,

F.A. tl9ltFI) Blwhcm,

J, 241, 71-32.

[.!I 81wn, R,W. #IId Tubin, A&J. /199#) FAJRR J. 5, bwv-I-ml,

IS) Sceburg, P. (I990) In: CIAUA tend 13cnxsdiWpinc Rwptsr ~ubt~p~~~~i~~~~, CJ. a&i Ccwt, E. cds) pp. W-XI, Rwm Prelr,, NW Vurk, &I M~~hler, H., S/twl, FL, hlttlhsrbr, P., Rlch#td~, J&i., PrrwW C.. Mottrns, f. tint1 Ifwtkc, b. (1991) in: Trrnrmittrr hmlnu held Rwcprerr Tranrductian tend M~drlr for Drug Rovclupmrnt (Crrrta, E, rmd Barnard, EA. cds) Ruw Pws, NW Ycwk tin prcrxl, i7) Kitknerx, ELF. and furncr, A.J. (I9rlil) liwhcm. f, 256, 291-294. [li) Bunyan, M,J. ttnd Stcphcnsan, F.A, (1989) J. Nwaehcm. 53, I52- IN. (91 SUIO, T.N. and Nettl, J.H. (1989) J, Neurouhem, 53, lO89-1095. [IO] %[email protected], E.A#, Bugpan, M.1, tend C’a%tleni, S,O. (1989) ~W3Ei betto 243, 35%362, [tl] Stcphcnaan, P.A., Dugpn. M,J, and Pallrrd, S, (1990) J. Uiol. c3lcnr~ 265, 21160-21 L65. 112) ESrnkc, CL, Mcrtenr, S., Trctxiwk, A,, Gillex-rcn, R. and Mehlcr, #, (1990) Eur. J. Phnrmacsl. 189, 337-340s [13) Bcnkc, IX, Mcrtcns, S., Trerriak, A,, CiiliwWt, 13. nnd Malrlcr, H, (1991) J. Viol, Chem. 266, 4478-4483# [ 141 Rdnkc, D., Mr‘rtcnr, S., Cicin.Snin, A. and Mehlcr, l-l. (1991) J, Rcrsp. Rcr. (In prcxa),

May

1991

(I $1 iRCiIkPr, t%, MetWn~, 1.. l’f~‘~ibl8k.A., 6lllaxrcn, la. #RBhluhlsr. tl. tlW1 (rubmlrrcd).

[Id] Ewwl, M,, Shlvcrs, 0.13., bilddens, tl., Muhlcr, H, and ?ierbrrry, PI. CIW) J. ‘.%I Dial, 110, IflJJ-IMHI. 1171 Fuieht, K., Adamikcr, D, and Jic:lharl, W. (1989) IX%4 bat. 261, f2-H, [I%1 LBtldcnr, W., Prhehctr, U.B., KWw, hl., Klllirch, I., KelnPncn, K., Mtwycr, Cl.. Sprrnyel, R. and !Qekurg, P&l. (IWO) Nature 346, 648mtSl. tl9) Sl$d, E., Blur, R., Trubc, CL. Mehlcr, W. and M&lbcrbc, P, (1990) Ncurtln 5, 703-711, t2fll VWdWarn, ‘~.A,, Rrn~uhn, A., Ymcr, S., Seeburg, P,H, and Sakmann, ES, (1990) Ncura~~ 4, 919-928. 1211 %n& S..3. (1973) P. Am. Chcnr, Sac. 95, 132%1333, 1221 Athcrton, E. and Sheppard, R.C, (19117) in: The Pcptidec: hnalyix, Synthrrlx, Blalafly, vol. S (Wdcnfrisnd, S, and Mclcnhafcr, J. eds) pp. l-31, Acrdcmic Prw, NW York. 1231 Skttlli, O., Roprax, P., Trexeiok, A., Eh~ronrna, G,, Gillcrrtn, 11. turd Cabbiani, 0, [l9W) I, Cell Dicrl. 103, 2787-2796, (241 Ilrttnd, E,,Altnmnn, A..Grllnrfrld, M., Ulmcr, A&l. and Fiwl, Cf.-B. (1986) Immunebierlegy 172, 33-36, 1251 &ha&, P. nnd Mshlcr, H. (1983) Eur. J, Ph#rmasol, US. 323-324. 1261 Richards, JG., Mohlcr, W. and EltICfely, W. (1986) in: Nrurahiaerlremistry: Modern Mcthadx and Applieationx, pp. 629-677, Alan R, Lisx, NW York.