Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C

Volume 27g, number ;I, 222-~4

FEBSOg)6t

J.nunry 1991

,~ I",~l F~der~d~n of l~uro~,~fl l~io~hemi~al $o~lelle~ 0OI4J7~}~91/$.t,~10 HDONI~ 0014;1791910001~J

Expression of the GABAB receptor in Xenopusoocytes and inhibition of the response by activation of protein kinase C K o h t a r o T a n i y a m a ='*, Koichiro T a k e d a ~, Hiroshi Andcg, Takayosi K u n o ~ and Chikako T a n a k a ~

Depc~rmmmspf ~Phem.acoiolu mul aPh~slal~lly, Kob¢ Universit.~ $¢h~al of Medh i~e, Kob¢ dSO J~pmt Recetved 2[~ October 1990; revised verston received6 December 1990 The functional G A B A I re¢eplor was ¢tcpresscd ht Xenopl~roocytes by in,~hng mRNA obtained from the ¢¢rcbellum ol' the ra~, Apphc~hon o GABA in the presence of bic,iculhn© reduced ~ hyperpolarD~hon 0nder current.clamp conditions and an outward c;urr~nt under volt.lie.clam I ¢onddions. Baclofcn mimicked the effect o r G ^ B A in |he prcsenceof b=cuculhn¢, and the effect or ba¢lol'¢n was antagonized by ph=clofcn rh, GABA.mdueed outward current was shghtly =nhiblted by trea',rnent w=|h GDP.fl.S and was complelel), mhib=tcd by treatment wtth GTP./,$, Th, activatm.t of protein km=tse C by 12.O.tctradeeanoylphorbol.l:l.=cctaze (TPA}, but not 4.x,phorbol-12,13-dtdccano.te. suppres~d the O ABAR re ceptor.mcdlated hyperpolarlzatlon, ned the effect or TPA was antagonized by sphmllosm¢, Thus, |tchvatlon of prolem ktaasc C mlnbits the =~ pressed GABA~ r¢~,eptor.medtatcdresponse GABAR receptor, mRNA rejected oocyte, Protein kmas,=C

I, I N T R O D U C T I O N The ~-aminobutyric acid (GABA) receptors have been classified ,nto two subtypes, termed GABAA and GABAa receptors, on the basts of their pharmacological properties [3,4]. The GABA^ receptor wttl~ its integrated CI" channel is now well characterized and recent studies revealed the amino acid sequence of this receptor [16,22]. GABAu receptors have been proposed to be coupled to Ca = ÷ channels in dorsal root gangha [8,11] and to K" channels in hlppocampal pyramidal cells [2,9]. The A'enopus oocyte is a useful model to examine the molecular mechamsms o f the modulauon of neurotransmitter responsed and ion channels. The GABAA receptor has been expressed in Xenopus oocytes by rejecting RNA from the rat brain [12] and the chick brain [17,25] and retina ill, and the GABAA receptor-mediated response was found to be suppressed by the actwatmn of protein kmase C [18]. On the other hand, the expresslon of the GABAB receptors m the Xenopus oocytes by injecting RNA has not been reported. The concentration of G A B A s sites in the cerebellum has been shown to be h~gher than elsewhere m the rat brain, as determined by receptor autoradiography [27]. We injected m R N A from the rat cerebellum and examined the propertie~ of the GABA~ receptor m relation to mtracellular signal transductton m Xenopus oocytes.

2, M A T E R I A L S

AND

METHODS

Total R.NA was extracted from th¢~cerebellum of 28 day old r= using a iIuan~dln¢ th~ocyanale/phenollchloroform procedure iS' Poly.A ° R N A (mRNA) was purified by passag(=through an ollgo (dl cellulose affintty column Tile oocytes from Xenopu~ laews v,er defolhculated by (remmcnt wL~h collr, genase Each defolliculale oocyte was mjccIed v,llh 50 nl of m R N A solut=on(l mglml tn water' and incubated for 2-"/da!,s at Ig"C tn sterileB:srth'ssolutmn (Nat 90 7 raM. KCI I raM, N a H C O ~ 2.38 raM. MgSO, 0 81 raM, Ca(NOr. 0 34 raM, CaCI: 0 41 raM, Hepes/NaOH 9 | raM, pH 7.6) wt|h 0 mg/ml gentamycln and 50 U/ml nystahn A single oocyte was place m a chamber and perfused with medium composed of NaC190 7 mV, KCI ! raM, NaHCOt 2 38 raM, MgSO.~ 0 81 raM, CaCI= 0 82 mlV Hepes/NaOH l0 raM, pH 7 6, at room temperature The oocyte w~ tmpaled w=th two m=croelectrodes, and responses =o substances wet re~orded using current-clamp or voltage-clamp c=rcuns The follo~ mg chemicals wcre obtmned from the LndLcatedfirms "r-ammobutyr~ acsd (GABA), blcuculhne methlodlde, GDP./3.S, GTP--r.S, 12.d tetradecanoylphorbol-13.acetate (TPA) and 4c~-phorbol-12,13-d decanoate (4c=.PDD) (Sigma), baclofen (C~ba Gelgy), phaclof¢ (Tachs) and sphlngoslne (Serdary Research LaboratoneO

3, R E S U L T S AND D I S C U S S I O N

*Present address The 2nd Department of Phrrmacology, Nagasakl Umverslty School of MeWcme, Nagasakl 852, Japan

The effect of GABA was usually tested in th presence of bzcuculline, to avoid the GABAA receptoz mediated response. Under current-clamp condmon., the apphcation of GABA m the presence of bicuculhn induced a hyperpolarization in the oocytes injected wit R N A derived from the rat cerebellum (Fig. IA), but nc in the uninjected oocytes or m oocytes rejected wit medium containing no RNA. The GABA-induce hyperpolarizatton occurred in 5 of 88 oocytes, 7 of 6 oocytes, 7 of 71 oocytes and 0 of 41 oocytes 3 days, days, 5 days and 7 days after rejecting RNA, respectw(

222

Pubhshed by E/sewer Science Pubhshers B V (Blomedtcal Dwmo

Correspondence address C Tanaka, Department of Pharmacology, Kobe Umverstw School of Medicine, Kob¢ 650, Japan

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ly. In the absence of btcucullme, some RNA injected oocytes from the same donor showed a depolarizing response of GABAA receptor origin. GABA generally depolarizes membranes of oocytes rejected w~th RNA from the brain [12,17,25] and retina [1], which is mediated by the stimulation of the bicuculline=sensitive GABAA receptor. Under voltage-clamp condtt,ons, the application of GABA in the presence of bicuculline induced an outward cmrent at the holding potentlal of -40 mV, however at the holding potential of -70 mV OABA failed to induce any current (Fig. IB), thereby suggesting that the K ~" channel was opened by stimulation of the GABAn receptor. Baclofen, a GABAB receptor agonist mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagomzed by phaclofen, a selectlve GABAB receptor antagonist (Fig. IC). These results indicate that the functional GABAB receptor is expressed in oocytes when RNA from the rat cerebellum ~s reJected rote their tissues In the dorsal root ganghon ceils, stimulation of the GABAs receptor decreases the Ca z + current [7,21]. In the hippocampus, the OABAB receptors are located

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Time oo,,rse (mini Fig 3 Suppresslon of GABA-mduced hyperpolanzatlon by actwat~on of protem k,nase C, unde~ current.clamp co~dmons BIcuculhne at 10- ~ M was present m the perfuslon medmm. TPA, 4¢~-PDD and sphmgosme were apphed 10 ram, 10 mm and 15 mm before the aad,tlon of GABA

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both pie. and postsynaptically, Eilher baclofen or G A B A in the presence of bieueulline incretses K " con. du¢tance and induce~ a hyperpolarizatlon o f pyramidal cell mombranes due to the stimulation of the po~tsynap. tic GABAr~ receptors [9, tO, 14,19], In the hippocampu=, the response mediated by the activation o f postsynaptic GA]BA~, but not presynapti¢ G A B A ~ receptor i~ phaciofen.sensitive [9], The GABA-tnduced outward current was slightly inhibited by treatment with GDP-dS, the non-hydrolysablc analogue of G D P and was completely inhibited by treatment with G T P . ~ $ , the non-hydrolysable analogue o f GTP (Fig, 2), thereby indicating that the expressed G A B A n receptor is coupled to G T P - b i n d i n 8 proteins, A phorbol ester, 12-O.tetradecanoyl.phorbol.13.ace. tate (TPA) suppressed the GABAB receptor.mediated hyperpolarlzation in the oocytes injected with R N A (Fig, 3), in contrast, 4~-phorbol.t2,13-didecanoate, a n o n . a c t w a t i n 8 analog of T P A , h a d no effect on the G A B A a receptor.mediated response. The suppressing effect of T P A was antagonized by sphingoslne, a com. p o u n d which inhibits the activity o f protein kinase C. These results indicate that the response mediated by stimulation o f the expressed G A B A ~ receptor is suppressed by the activation o f protein kinase C , corresponding to the findings m the hippocampus, in which the activation of protem klnase C by phorbol ester suppresses the responses mediated by the stimulation o f either pre- or postsynaptic G A B A a receptors [t21. Protein klnase C may act through phosphoryiation o f specific proteins T h e mechanism underlying the suppresston o f the expressed G A B A ~ receptor-medzated response in the o o c y t e requires further study. Various voltage-dependent and r e c e p t o r - m e d m t e d 1on channels were found to be m o d u l a t e d by the activation of protein kinase C [23]. It has been shown that the a c t i v a u o n o f protein kinase C m o d u l a t e s nauve ion channels o f intact folhculated oocytes, for example, the adenosineinduced K ÷ channel [6], a n d the expressed ton channels of the R N A injected oocytes, such as voltage-dependent C a 2. channel [15,24], N a ~ channel [241 a n d the GABAA r e c e p t o r - m e d i a t e d C I - channel [24]. 5 - H y d r o x y t r y p t a m i n e - , G A B A - and acetylchohne (ACh)-induced inward currents were s h o w n to be suppressed by actwatzon of protein kinase C [18]. Activation o f protein k m a s e C was f o u n d to p h o s p h o r y l a t e receptors such as the nicotinic A C h receptor, ~3a d r e n o c e p t o r , c~t-adrenoceptor, a n d the ¢x-subunit o f G T P - b i n d i n g p r o t e i n (GO, a n d the N a * channel [13,20]. T h e G A B A n receptor is the m o s t plausible target protein o f protein kinabe C because a c t w a t i o n o f the e n z y m e seems to p r o m o t e a f o r m o f heterogeneous desensitization [13]. It zs also possible that the G T P binding p r o t e i n is a target of protein kinase C. T h e r e are multiple subspecies o f protein kinase C, and at least 4 of them a , BI, ~ I I and .~ are present in the brain [20]. 224

January IWl

We could not determine which sgbtpe¢ies of protein kinase C participates in inhibition o f the G A B A ~ receptor.mediated response, since we did nor rule out the possibility that the m R N A from the ,at cerebellum used in our study may have contained m R N A encoding th.: multipl~ lubspecies o f protein klnase C [20,26] together with that encoding the GABAN receptor, ArlcnowIedzemem, Thh work wa~ supported by ilrtmtt from the

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