Regulation of GABAA receptor function by protein kinase C phosphorylation

Neuron, Vol. 12,1081-1095, May, 1994, Copyright 0 1994 by Cell Press

Regulation of GABA* Receptor Function by Protein Kinase C Phosphorylation

Belinda J. Krishek,* Xinmin Xie,* Craig Blackstone,+ Richard 1. Huganir,+ Stephen J. Moss,* and Trevor C. Smart* *Department of Pharmacology The School of Pharmacy 29-39 Brunswick Square London WCIN IAX England +Howard Hughes Medical Institute The Johns Hopkins School of Medicine Baltimore, Maryland 21205 *IRC Institute for Molecular Cell Biology University College Gower Street London WCIE 6BT England

Summary

CABA* receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of p and y2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in pl and y2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the y2 subunit exerted the largest effect on the CABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.

Introduction

y-Aminobutyric acid (GABA) is recognized as the major inhibitory synaptic neurotransmitter within the brain causing the activation of type A GABA (GABA*) receptors (Olsen and Tobin, 1990). Although the pre- cise composition of a native GABAA receptor is un- known, molecular cloning studies have revealed a wide diversity of individual GABAA receptor subunits. By comparing deduced amino acid sequences, these subunits are divided into five discrete families: al-6, bl-4, ~1-3, 61, and ~1-2 (Schofield et al., 1987; Burt and Kamatchi, 1991). On the basis of comparative ho- mology, GABAA receptors are designated as members of a superfamily of l igand-gated ion channels that includes nicotinic acetylcholine receptors and glycine receptors (Barnard et al., 1987). Members of this super-

family are proposed to share a common transmembrane topology consisting of a large extracellular N-terminal domain, four membrane-spanning regions, and a large intracellular loop region between transmembrane regions 3 and 4 (Barnard et al., 1987). Additional diversity in the structure of GABAA receptors is generated by alternative splicing of the ~2, p2, and p4 subunit mRNAs (Whiting et al., 1990; Kofuji et al., 1991; Bateson et al., 1991; Harvey et al., 1994). Functional studies of GABA* receptors have routinely utilized heterologous expression of three GABA* receptor subunits (a, P, and y), either individually or together, to produce functional GABA-gated chloride channels (Schofield et al., 1987; Blair et al., 1988; Burt and Kamat- chi, 1991).

An important means of regulating the function of l igand-gated ion channels is to modify receptor structure covalently via phosphorylation (Swope et al., 1992; Raymond et al., 1993). Consensus sites for phosphorylation are found within the large intracellular domains of many GABAA receptor subunits. For example, all b subunits (01-4) encode a conserved site for phosphorylation by protein kinase A (PKA; serine S409 in the 01 subunit; Ymer et al., 1989; Swope et al., 1992). Moreover, the alternatively spliced versions of the y2 subunit, termed short (~2s) and long (y2L), differ by only an extra 8 amino acids in the long form (Whiting et al., 1990; Kofuji et al., 1991), which contains aconsensussequenceforphosphorylat ion byprotein kinase C (PKC; Moss et al., 1992a; Whiting et al., 1990). Interestingly, mRNAs encoding these two forms of they2 subunit have differing patterns of temporal and spatial expression in the brain (Glencorse et al., 1992), suggestinganovel role for phosphorylation in regulating CABAA receptor function.

In accordance with these observations, affinity- purified preparations of GABAA receptors are substrates for PKA, PKC (Kirkness et al., 1989; Browning et al., 1990), and an unidentified kinase (Sweetnam et al., 1988). The relatively low abundance of GABAA receptors and their heterogeneity in the brain make any identification of the sites of phosphorylation difficult. To obviate these problems, the intracellular regions of GABAA receptors have been expressed as Escherichia coli fusion proteins. The intracellular domain of the PI subunit is phosphorylated on S409 by both PKA and PKC (Moss et al., 1992a), whereas the intracellular domains of the y2S and y2L subunits are phosphorylated by PKC on S327, and phosphorylation of the y2L subunit is on S343, within the alternatively spliced region (Whiting et al., 1990; Moss et al., 1992a). Furthermore, GABAA receptors may also be substrates for tyrosine kinases, as both the yl and y2 subunits contain suitable consensus sequences for tyrosine phosphorylation (Pritchett et al., 1989; Swope et al., 1992).

P1: CtlF

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C 12 D 12 Figure 1. immunoprecipitatiofl of CABAh Receptor Subunits from Transiently Transfected A293 Ceils

(A) A293 ceils were transfected with equimolar mixes of algl

4200 4200

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(ianel) or a1/31y2L (lane 2) expression constructs. After 48 hr, cells were labeled with [%]methionine Kl.25 r&i/ml) for 4 hr. ThecelIswerethenlysed,andtheGABAhreceptorsubunitswere purified by immunoprecipitation using affinity-purified anti-g? antiserum coupled to protein A-Sepharose. Lmmunocomplexes

PI: - were resolved by SDS-polyacrylamide gel electrophoresis fol-

(Xl : ;r lowed by fluorography. Lane 3, control transfected cells. Molecu-

446 446 lar masses of marker proteins are indicated on the right. (B) Ceils were transfected and labeled with [%]methionine as

430 430 described in (A). GAB& receptor subunits were immunoprecipitated using affinity-purified anti-al antiserum coupled to protein A-Sepharose. Lane 1, cells expressing alpI subunits; lane2, cells expressing alply2L subunits; lane 3, cells expressing alfily2L subunits in which, prior to immunoprecipitation, theanti-al an-

tiserum had been preadsorbed with 50 Kg of the peptide used for immunization. (C) A293 cells expressing alply2L subunits were labeled with [‘%]methionine. A cell lysate was prepared under denaturing conditions, and the al subunit was immunoprecipitated using anti-al antiserum as detailed in (B) (Lane 1) or anti-al antiserum that had been preadsorbed with 50 Lrg of the peptide used for immunization (lane 2). (D) A cell lysate was prepared under denaturing conditions from A293 cells expressing alply2L subunits. The bl subunit was then isolated using anti-61 antiserum as detailed in (A) (lane I) or anti-81 antiserum that had been preadsorbed with 50 bg of the fusion protein used for immunization (lane 2).

Some of the functional effects of GABAA receptor phosphorylation have been examined in native neurons and cell expression systems. These effects are complex and sometimes apparently contradictory. For example, GABA-activated responses can be inhibited by phosphorylation (Porter et al., 1990; Moss et al., 1992b), whereas in some neurons, a continuous “rundown” of the GABA response amplitude can be prevented by agents known to promote phosphorylation (Stelzer et ai., 1988; Gyenes et al., 1988; Stelzer, 1992). These differential effects of phosphorylation may result from heterogeneity in GABAA receptor structure, but interpretations are often complicated by the nonspecific effects of many drugs used to modulate kinase activity (Leidenheimer et al., 1991).

We have used a combination of biochemical and electrophysiologicai techniques to study the functional consequences of phosphorylation of native neuronal and recombinant GABA* receptors by PKC.

These results demonstrate that PKC phosphorylation has differential effects depending on the GABAA receptor subunit composition and the number of phosphorylation sites contained within these subunits.

Results

Phosphorylation of the PI, ?2S, and y2L GABAA Receptor Subunits by PKC We utilized the transient expression of GABAA receptor subunits inA293cells(Pritchettetal.,1989;Mosset al., 1991,1992a) to examine phosphorylation of CABAR receptor subunits by PKC. Transfected cells expressing al81 or alply2L subunits were labeled with [35S]methionine prior to immunoprecipitation of the subunits with specific antisera. Using affinity-purified antiserum against the PI subunit under native conditions, the al subunit migrated as a 52 kDa protein whereas the fil subunit migrated as a 58 kDa protein,

GABA, Receptors and PKC 1083

A 12 34 56 78 910

- + - + - + - + - +

B 1 2 3 4 5 6

PKCa + + - + + +

PMA + + + + +

PC al+

with a putative proteolytic breakdown product at 56 kDa(FigurelA, lanes 1 and 2; Moss et al., 1992a, 1992b). Native immunoprecipitation with anti-al antiserum gave similar results (Figure 16, lanes 1 and 2). Under denaturing conditions the anti-al antiserum precipi- tated the 52 kDa band in addition to bands of 48 and 50 kDa (Figure IC, lane 1). The lower bands presumably represent degradation products of the 52 kDa band. lmmunoprecipitation of these bands was blocked by preadsorption of the immunizing peptide (Figure IC, lane2). Utilizing the anti-81 antiserum under denaturing conditions resulted in the immunoprecipitation of just the 56 and 58 kDa bands (Figure ID, lane 1). The 56 and 58 kDa bands were specifically phosphorylated by PKA, providing additional evidence that these bands represent the 81 subunit (Moss et al., 1992a, 1992b). In contrast, the y2 subunit was difficult to de- tect biochemically, appearing to be highly sensitive to proteolysis and migrating as a diffuse band at 42 kDa using either the anti-al or anti-B1 antiserum; this is in agreement with earlier observations (Moss et al., 1991a; Hadingham et al., 1992). Utilizing antibodies against the ~2 subunit, previously raised against an E. coli fusion protein of the large intracellular domain of ~2, yielded similar results even though this antiserum was capable of immunoprecipitating rH]fluma- zenil-labeled GABAA receptors from mouse brain (data not shown).

To examine the phosphorylation of GABAA receptors by PKC, transfected cells were prelabeled with [32P]orthophosphoric acid. GABAA receptor subunits were then immunoprecipitated using the anti61 antiserum under native conditions. Whereas the l31 sub-

Figure2. Phosphorylation of CABAA Re ceptor Subunits Expressed in A293 Cells

(A) A293 cells expressing recombinant GABA* receptors composed of alp1 (lanes 1 and 2), alBly2L (lanes 3 and 4), alBl(S409A) (lanes 5 and 6), alBl(S409A)y2L (lanes 7 and 8), or control untransfected cells (lanes 9 and IO) were labeled with 12P (0.5 mCi/ml) for 4 hr. The cells were then treated with (+) or without (-) 50 nM PMA for 15 min and lysed. GABA, receptors wereimmunoprecipitated using anti-Bl antiserum coupled to protein A-Sepharose. The immunocomplexes were then resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. (B) A293 cells expressing GABA, receptors with (+) or without (-) PKCa composed of al81 + PKCa (lanes 1 and 2), alPI - PKCa (lane 3), alBly2L + PKCa (lane 4), or alBl(S409A) + PKCa (lane 5), or untransfected cells (lane 6) were labeled with n2P as described in (A). Transfected cells were treated with (+) or without (-) 50 nM PMA for 15 min. GABAA receptor subunits were isolated by immunoprecipitation and analyzed as in (A).

unit showed low levels of basal phosphorylation, phosphorylation of the al subunit was not detected (Figure 2A, lanes 1 and 3). Treatment of cells expressing either the alB1 or alBly2L subunits with the PKC activator phorbol 12-myristate 13-acetate (PMA) dra- matically increased phosphorylation of the Bl subunit (Figure 2A, lanes 2 and 4). In contrast, the al subunit was not significantly phosphorylated. An identical pattern of phosphorylation of the l31 subunitwas seen on coexpression with the al and ~2s subunits (data not shown). The bands in Figure 2A (lanes 2 and 4) were excised and subjected to both phosphopeptide mapping and phosphoamino acid analysis. Phosphor- ylation of the 81 subunit occurred within a single phosphopeptide(Figure3A)exclusivelyon serine residues (Figure 3B).This peptide map is identical to phosphopeptide maps of a Bl subunit fusion protein encoding the entire intracellular loop between transmembrane regions 3 and 4 after phosphorylation by purified rat brain PKC (Moss et al., 1992b). Site-specific mutagenesis identified S409 as the major site of PKC phosphorylation in the fusion protein. Mutation of this residue to alanine (S409A) in the 81 subunit dra- matically reduced the level of phosphorylation after treatment with PMA when Bl was expressed with either the al subunit alone, or the al and y2L subunits (Figure 2A, lanes 5-8). These results identified S409 as themajorsubstrateof PKCwithin the81 subunitwhen expressed in A293 cells.

To increase the levels of PKC within A293 cells, GA- BAA receptor subunit cDNAs were cotransfected with a cDNA encoding the a isoform of PKC (SRa-PKCa; Kaibuchi et al., 1989). After treatment with PMA, GA-

Neuron 1084

al pyzL - -k=

B

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THR +

TYR -e

ELECTROPHORESIS

a1 p1 a1 ply2L

Figure 3. Phosphopeptide Mapping and Phosphoamino Acid Analysis of GABAA Receptor Subunits (A) The phosphorylated PI subunits from A293 cells (shown in Figure 2A, lanes 2 and 4) were excised and digested with trypsin (both the 56 and 58 kDa bands). The resulting phosphopeptides were then subjected to phosphopeptide mapping and visualized by autoradiography. Identical results were obtained from cells coexpressing GABAA receptor subunits and rat PKCa. (6) The phosphopeptides produced from the PI subunits (in Fig- ure 2A, lanes 2 and 4) were hydrolyzed with 6 N HCI, and the resulting phosphoamino acids were separated by electrophore-

5uM GABA _ - -

0 a101yzL Controi I * oi0lyZL + PDBu 1

Figure4. Effect of PDBu on GABA-induced Currents in A293 Cells Transfected with alply2L cDNAs (A) Whole-cell recordings of CABA-induced membrane currents after rapid application of GABA (5 PM) to A293 cells voltage clamped at -50 mV in theabsenceand presenceof 500 nM PDBu. The records were obtained at various times (t, minutes) after formation of the whole-cell configuration, and CABA was applied for the duration indicated by the solid line. (B) Stability plot of the CABA-activated current amplitude (5 PM GABA; 100% defined at time zero) versus whole-cell recording time in the absence and presence of 500 nM PDBu. All points are means + SEM from 5-11 cells.

BAA receptors were isolated by immunoprecipitation. Coexpression of PKCa with the al and fil subunits resulted in a 2-to 3-fold increase in phosphorylation of only the /3Z subunit as compared with experiments withoutcotransfection (Figure25,lanes2and3). Phos- phorylation occurred only on serine residues within a single phosphopeptide identical to the pattern of phosphorylation observed without cotransfection with PKCa. Mutation of S409 in the 01 subunit elimi- nated most of the phosphorylation (Figure 25, lane 5). The PI subunit was phosphorylated in an apparently identical manner when expressed with the y2L subunit on coexpression with PKCa. Phosphorylation of the y2L subunit could not be detected, presumably owing to the susceptibility of this subunit to proteolysis (Figure 2B, lane 4). Similar results were obtained

sis. The positions of phosphoserine, phosphothreonine, and phosphotyrosine standards are indicated. The PI subunits isolated from ceils coexpressing PKCa were also phosphorylated only on serine residues.

CABAA Receptors and PKC 1085

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Figure 5. Effect of PDBu on GABA-Induced Currents in A293 Cells Cotransfected with GABAA Receptor cDNAs and SRa-PKCa DNA

(A) GABA-activated currents recorded at various times from A293 cells expressing alPI (upper trace) or a’lf31(%09A) (lower trace) receptor constructs and PKCa. PDBu (500 nM) was included in the pipette solution. The plot shows the stability of the relative amplitude of GABA-activated currents for wild-type receptors in the absence and presence of PDBu and for mutant receptors exposed to 500 nM PDBu. The records were obtained at various times (t, minutes) after formation of the whole-cell configuration, and GABA was applied for the duration indicated by the solid line. (B) CABA-activated currents recorded from cells expressing alply2S (upper) or alf3l(S409A)T2S(S327A) (lower) GABAn receptors exposed to intracellular PDBu. The stability of the GABA-activated current relative amplitude is shown in the right-hand plot for wild-type receptors in the absence and presence of PDBu and for mutant receptors exposed to PDBu. (C) GABA-induced currents recorded from A293 cells expressing algly2L (upper) or alL31(%09A)r2L(S327A,S343A) (lower) receptor constructs with intracellular PDBu. The stability plot includes wild-type receptors, in the presence of PDBu or a-PDD and in the absence of phorbols; the mutant receptor was exposed to PDBu. Holding potential in all cells was -50 mV.

NWKNl 1086

with they2S subunit. However, previous studies using fusion proteins encoding the large intracellular regions of the y2S and y2L subunits have identified phosphorylation sites for PKC. Both forms of this subunit are phosphorylated on a common residue, S327. In addition, they21 subunit is phosphorylated by PKC on 5343, which is located within an 8 amino acid insertion created via alternative splicing (Moss et al., 199213). The assembly and functional expression of murine heteromeric GABAA receptors consisting of al, 81, and ~2s or y2L subunits have been previously assessed by radioligand binding and pharmacological analyses (Moss et al., 1991, 199213; Smart et al., 1991).

Effect of Phorbol Esters on GABA-Activated Membrane Currents: Human Embryonic Kidney Cell Expression Studies Heteromeric GABA* receptors incorporating al, 61, y2S, and y2L subunits were expressed in A293 cells to assess the functional consequences of phosphorylation of the PI and y2 subunits. The cells were subjected to whole-cell recording at a holding potential of -50 mV. When recording from cells expressing alpl, alfJly2S, or alfily2L receptor subunits, the GABA- activated responses evoked by rapid applications of 5 uM GABA from an adjacent multibarrelled pipette generally did not exhibit significant rundown for up to 35-60 min following formation of the whole-cell recording mode (92% k 9% of the initial response to GABA; n = 45 cells; Figure 4). The effect of PKC phosphorylation on GABAA receptor function was ad- dressed by activating PKC using phorbol esters. These agents were included in the intracellular pipette solution, thereby limiting exposure to just the recorded cell. Under these conditions, in cells expressing alpl, a1(31y2S, or alPly2L cDNAs, 500 nM phorbol 12,13 dibutyrate (PDBu) produced no significant change in the peak GABA-induced current amplitude after 20- 30 min following initiation of whole-cell recording (Figure 4). The lack of a clear effect of the phorbol esters could reflect a dilution of PKC by intracellular dialysis. To overcome this potential problem in A293 cells, we increased the levels of PKC by cotransfecting cDNAs for the GABAA receptor subunits with SRa- PKCa cDNA. Cotransfection was previously demonstrated to increase the level of phosphorylation of the 81 subunit (Figure 2). Using this protocol, the peak amplitudes of GABA-activated currents recorded from the wild-type receptors formed from al81, a1[31y2S, and alply2L were reduced by up to 45% over IO-50 min following intracellular application of 500 nM PDBu (Figure 5). Responses recorded from GABA* receptors containing the ~21. subunit were inhibited to a greater extent by phorbol esters (32% mean inhibition) compared with responses trans- duced via y2Scontaining receptors (22% inhibition, n = 9; Figures 5B and SC). Control recordings using a pipette solution without PDBu from cells cotransfected with GABA* receptor cDNAs and SRa-PKCa

cDNA revealed little if any rundown in the GABA- activated membrane current.

The specificity of action of the phorbol esters was ascertained by either using a phorbol ester that does not activate PKC, e.g., 4a-phorbol 12,13 didecanoate (a-PDD), or recording from cells expressing mutant forms of the GABAA receptor subunits in which key serine residues on /31 (S409), y2S (S327), and y2L (S32;I, and S343), previously demonstrated to be substrates for PKC (Moss et al., 1992a), are replaced by nonphosphorylated alanine (A) residues. jntracellular application of a-PDD (500 nM) to cells coexpressing PKCa and alply21. did not affect the amplitude of the GABA- induced current, suggesting that the action of PDBu was mediated by the activation of PKC (Figure 56). This conclusion was supported by the lack of effect of intracellular PDBu on GABA-induced currents recorded from cells in the absence of cotransfected PKCa and on GABAh receptors from cells expressing PKCa and the following mutated GABA* receptor constructs: alpI (S409A), alL3l(S409A)*{2S(S327A), or alfil- (S40VA)r2L(S327A,S343A) cDNAs (Figure 5). Control recordings from cells transfected with these mutant constructs and not exposed to PDBu exhibited weli- maintained responses to GABA over 30-45 min.

CABA-Activated Membrane Current Kinetics The effects of PKC-mediated phosphorylation on the kinetics of CABA-induced membrane currents were examined by rapidly applying GABA to A293 cells expressing wild-type or mutant recombinant GABAx receptors and PKCa. Cells expressing GABAA receptors comprisingaID subunitsusuallyexhibitedabiphasic decay (Figure 5A) that could be described by two exponential time constants (rfast, 2.89 + 0.38 s; zslow, 28.9 + 3.3 s) and may represent underlying receptor desensitization. PDBu (250 nM) reduced the fast phase of apparent desensitization (see Figure 5A) such that the residual currents were best fitted with a single exponential (41.1 5 12 s). This gave the appearance that desensitization was slowed, but the majority of the inhibition was accounted for by the loss of the fast phase, with little effect on the time constants for decay. For the $&containing GABA), receptors, single time constants sufficed to describe most of the current decays (al@ly2S, 31.85 + 16 s; alply2L, 24.6 i 9 s), and these were relatively unaffected by phorbol ester treatment even though the current amplitudes were depressed (Figure 58). By mutating S409 in the f31 subunit,thedesensitization kineticsofalfll(S409A) GABAA receptors in the absence (1.45 k 0.86 and 26.3 f 11 s) or presence (1.37 -1- 0.26 and 24 + 14 s) of L3 phorbol esters were similar to those observed with the nonphosphorylated wild-type receptors (alpI). Similar mutations of serines at positions 327 (in y2S and y2L) and 343 (y2L) caused no obvious effect on the kinetics of presumed desensitization (see Pig- ures 5B and X). The results suggest that phosphorylation at these serine residues can affect the amplitude


A 1OpM GABA -40 ---y-- -

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Figure 6. GABA-Activated Membrane Cur- rents Recorded from Xenopus Oocytes In- jected with Various GABAA Receptor Con- structs

Xenopus oocytes were injected with alB1 (A), albly2L (B and C), and al~l(S409A)y2L (S327A,S343A) (D) GABAA receptor constructs. These receptors were exposed to bath-applied 250 nM PMA (A, B, and D) or 500 nM a-PDD (C). Records were made at varioustimesafterexposuretothephorbol esters (t, minutes). CABA was applied for the duration indicated by the solid bar. The membrane conductance was monitored

throughout by repetitively applying brief hyperpolarizing voltage command steps (-10 mV, 1 s, 0.2 Hz). Holding potential for all oocytes was -40 mV. The 50 nA calibration applies to (A) and (C). (E) Time course plot for the relative membrane conductance (control, 100%) induced by 10 PM CABA on CABA, receptor constructs alpI, alply2L, and al~l(S49A)~2L(S327A,S343A). PMA (250 nM) or a-PDD (500 nM) was applied at time zero. All points are means f SEM obtained from 15 different oocytes.

of the GABA-induced current, and in particular, phos- hibition, although the small initial enhancement in phorylation of S409 can reduce the proportion of fast the GABA-activated response was often present (Fig- desensitization. ure 6D).

Xenopus Expression Studies We investigated the action of PKC on recombinant GABAA receptors using the Xenopus oocyte to ob- serve whether the choice of expression system influ- enced the functional effects of PKC-induced phosphorylation on the GABAA receptor. Membrane currents evoked by bath-applied GABA to single Xen- opus oocytes were monitored after prior injection with cDNAs corresponding to alPI, alply2S, or al(31y2L CABA,+ receptor subunits. Under voltage clamp at -30 to -50 mV holding potential, the majority of injected oocytes responded to GABA (0.1 to 1000 vM) with inward membrane currents (IO nA to 1 PA) whose amplitudes were typically maintained over recording periods of up to 120 min. Subsequent bath application of 250 nM PMA often resulted in a small transient enhancement (5%-35% occurring within 3-5 min), yielding to a gradual decrease in the CABA-activated membrane current amplitude and conductance (Figure 6). However, inhibition of GABA-induced responses was not observed after exposure to the inactive a-PDD (500 nM; Figures 6C and 6E). Moreover, application of 250 nM PMA to the mutant GABAA receptors (alfil(S409A), alpl(S409A)y2S(S327A), and al~l(S409A)r2L(S327A,S343A)) did not result in any in-

Equilibrium Concentrat ion-Response Relationships Equilibrium concentration-responsecurvesfor GABA were constructed for all the aforementioned three subunit combinations by measuring the steady-state GABA-induced chloride conductance. These relationships revealed that the steady-state antagonism produced by prior exposure to 250 nM PMA for 30-90 min (Figure 6E) was of a mixed/noncompetitive type and was not readily reversible (Figure 7A). The maximum conductance increase induced by saturating concentrations of GABA (1 mM) was reduced with little change in the Hill coefficients or ECso values for GABA after exposure to PMA (Table 1). The percent inhibition plots for PMA demonstrated that the degree of depression of the GABA-induced responses was slightly dependent on the agonist concentration, consistent with a mixed inhibitory mechanism. Inter- estingly, the degree of inhibition was apparently dependent on the subunit composition of the receptor, asobserved with theA293cells(Figure8A).The inhibition induced by PMA appeared more effective with yZcontaining GABAA receptors, although the difference in sensitivities between receptorscontainingthe short and long forms of the ~2 subunit is smaller.

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Figure 7. Equilibrium Concentration-Response Curves for GABA-Induced Membrane Conductance Recorded from Oocytes Expressing Wild-Type or Mutant Receptors Membrane conductance (AGN; see Experimental Procedures) was recorded from wild-type (A) or mutant (B) receptors. All curves (control, open symbols; i-250 nM PMA, closed symbols) were obtained from 3-7 different oocytes for each receptor construct and have been normalized to the conductance induced by 10 PM CABA in control Ringer’s solution. The points represent means f SEM. Curves were fitted according to the logistic model (see Experimental Procedures). The concentration-response curves were measured after the effect of PMA had attained a steady state, usually 30 min following application. Response curves for the mutant receptors were also measured after a 30 min exposure to PMA.

Overall, the following rank order of inhibitory po- tency for 250 nM PMA was determined as

albly2L > alj3ly2S >> alfil.

The GABA concentration-response relationships for the mutant GABAA receptors were unaffected by PMA

(Figure 78). This indicated that the inhibitory effects of PMA were most likely mediated by PKC-induced phosphorylation of either the $1 or y2 subunits or both at the serine residues previously identified from phosphopeptide mapping and phosphoamino xid analysis. Current-voltage relationships for these GA-

Table 1. Analysis of the Equilibrium Concentration-Response Curves for GABA in Xenopus Oocytes Injected with GABAr Receptor cDNAs

EGO n

GABAA Receptor Subunit Composition Control +PMA Control +PMA

al)31 5.88 f 0.6 4.27 + 0.2 0.91 It 0.07 1.05 f 0.05 albly2S 10.03 + 0.21 13.01 + 0.41 ?.30 * 0.03 1.32 i- 0.04 albly2L 10.20 f 0.47 9.43 * 0.47 1.22 j 0.06 1.47 + 0.06 alpl(S409A) 8.30 ?r 0.77 7.40 + 0.37 0.77 * 0.04 0.83 ‘- 0.03 alpl(S409A)y2S(S327A) 10.06 i 0.38 10.65 f 0.45 1.07 + 0.04 1.10 * 0.04 alpl(S409A)y2L(S327A,S343A) 9.73 f 0.35 9.75 f 0.35 1.24 -c 0.05 1.20 * 0.04 alply2L(S327A,S343A) 10.13 i 0.34 9.74 f 0.65 1.22 f 0.04 1.04 + 0.06 alal(S409A)y2L 9.58 + 0.60 8.78 f 1.07 1.00 $ 0.05 0.87 it 0.07 alf%l(S409A)y2L(S327A) 10.93 + 0.47 11.82 ‘r 0.72 1.29 i- 0.06 1.34 * 0.09 alpl(S409A)y2L(S343A) 10.46 rk 0.39 14.14 + 0.45 1.35 i 0.06 7.71 f 0.07

The ECso and Hill coefficient (n) data were determined from fitting the logistic model to the normalized curves (see Experimental Procedures). All values are means f SEM.


Figure8. Percent Inhibition of GABA- Induced Conductance after Treatment with 250 nM PMA for Wild-Type and Mu- tant Receptors Expressed in Xenopus Oo- cytes

(A) Wild-type receptors; (B) selected mutant receptors. The points have been calculated from the data in Figures 7A and 9B, and the curves were generated using third or fourth order polynomials.

BAA receptor constructs revealed that the inhibition induced by the phorbol esters was independent of membrane voltage (data not shown).

Phosphorylation of S327 and S343 in ~21 Produces Differential Inhibition of GABA-Activated Current To examine the contribution of each of these phosphorylation sites in detail,we used site-directed mutagenesis to replace sequentially each serinewith an alanine. Four new CABAA receptor subunit constructs were assembled. Initiallythefunctional effect of phosphor- ylating the 2 serines (S327 and 5343) in y2L were studied in GABAA receptors comprising alpl(S409A)y2L. Application of 250 nM PMA induced a noncompetitive inhibition of the GABAconcentration-response curve (Figure 9). Phosphorylation of S327 and S343 was further analyzed by selective site-directed mutagenesis of each residue, yielding receptors with the following composition: alfil(S409A)r2L(S327A) and alPI (S409A)y2L(S343A). The former combination retains the serine residue in the alternatively spliced section of y2L while removing the serine normally present in both y2S and y2L. The latter construct retains the ser-

ine normally present in y2S and loses the serine in the alternatively spliced section. Expression of these receptor constructs in Xenopus oocytes resulted in functional receptors, with PMA (250 nM) causing inhibition of the induced GABA conductance. The degree of inhibition decreased with incrementing agonist concentration, and at high GABA concentrations (>I0 PM), membrane currents mediated by the alB1 (S409A)y2L(S327A) receptor constructs were more inhibited when compared with responses mediated by alPl(S409A)y2L(S343A) (Figure 9). Interestingly, the equilibrium response curves for the two forms of y2L subunit in thepresenceof PMA both exhibitedasmall lateral shift and depression of the maximum response (Figure 9). This type of inhibition suggests a complex mode of antagonism and is consistent with mixed inhibition (Smart and Constanti, 1986). For acomparison of the relative contribution of phosphorylation in the PI subunit for receptors comprising alply2L, we replaced both S327 and S343 in the y2L subunit with alanines and reverted to the wild-type 01 subunit, forming alfilr2L(S327AlS343A). These constructs mediated GABA-induced currents that were inhibited in

Figure 9. Equilibrium Concentration- Response Curves for GABA-Induced Mem- brane Conductance Recorded in Xenopus Oocytes from CABAn Receptors Compris- ing Selected Mutations in the PI and y2L Subunits in the Absence and Presence of PMA

(A) Comparison of the effect of mutating S409 in 01 with S327 and S343 in y2L. The broken line indicates the position of the curve for alpl(S409Ajy2L. Closed symbols, with 250 nM PMA; open symbols, without PMA. (B)Comparison of the 2 serines in y2L by selectively mutating 5327 or S343 while S409 in El remains mutated to alanine. The broken line indicates the position of the alpl(S409A)r2L(S343A) curve. The curves are normalized as described in Figure 7. All points are means f SEM and arefitted with the logistic model (see Experimental Proce- dures). Data were obtained from 3-8 oocytes per curve.

Neuron 1090

a noncompetitive fashion with a depression of the concentration-response curve and no obvious lateral shift (Figure 9). This type of depression was consistent with that observed with phorbol esters and GAB& receptors composed of only wild-type alB1 subunits. The percent inhibition plots indicated that the phosphorylation at S327 and S343 in y2L produced a larger inhibition of the GABA-activated response compared with phosphorylation at S409 in fil (Figure 88). This was most noticeable at lower GABA concentrations (<IO0 PM). Moreover, phosphorylation of S343 appeared to have agreater modulatory influence, partic- ularly at higher GABA concentrations (>I0 vM), compared with phosphorylation at S327. Phosphorylation at both S327 and S343 did not produce an addi- tive depression of the GABA concentration-response curve, since the inhibition of responses mediated via al~l(S409A)y2L was only slightly greater than that observed with alpl(S409A)r2L(S327A) (Figure 9). Thus the two sites on the y2L subunit may be mutually ex- clusive with regard to function. However, the amount of inhibition exerted by phosphorylation of the GA- BAA receptor 01 and ~2 subunits appears to be satura- ble, since no further depression in the GABA concentration-response curve could be achieved when applying phorbol esters to receptors comprising algly2L compared with alpl(S409A)y2L. Thus the extra serine at position 409 in the 01 subunit did not enable a further depression in the GABA dose-response curve.

Neuronal GABA-Activated Currents Are Inhibited by Phorbol Esters In comparison with the recombinant receptors, we examined whether a similar modulation of GABA- activated membrane currents by PKC-induced phosphorylation alsooccurred in native neurons. Recording from sympathetic ganglion neurons in primary dissociated cultures using whole-cell voltageclamp yielded mono- or biphasically decaying membrane currents after the rapid application of GABA (Figure 10). These responses are sensitive to inhibition by zinc and potentiation by benzodiazepines, suggesting that at least two different types of GABAA receptor constructs are present (e.g., receptors containing a y subunit and receptors lacking a y subunit; Draguhn et al., 1990; Smart et al., 1991). The GABA-induced responses were quite stable for up to 60 min without significant rundown in the response amplitude. Inclusion of 500 nM PDBu in the patch pipette solution led to a reduction in the response amplitude; however, in other neurons, this inhibition was not induced by the inactive phorbol a-PDD (500 nM; Figure 10). For neurons exhib- iting a biphasic decay of the GABA-activated current @fast, 1.47 + 0.7; f510W, 12.4 + 5 s), PDBu induced a reduction in the fast component of desensitization such that the residual current was fitted by a single exponential (27.7 + 16 s). In the presence of a-PDD, there was no effect on the current profile nor the time

A 5@4 GABA t0 I30 -- r- -

/

+PDBu

‘i)” 4

5pi\n GABA -

+aPDD

‘f

~ 0 Conriol ,A + a-PDD

c + PDRU

Figure 10. Effect of PDBu on GABA-Activated Membrane Cur- rents Recorded from Cultured Sympathetic Neurons

(A) CABA-activated current after rapid application of 5 +M CABA at time zero and at 30 and 50 min after the start of the whole-cell configuration (upper trace). Intracellular addition of 500 nM PDBu to a different neuron resulted in a declining GABA- activated current (middle trace). The inactive phorbol a-PDD (500 nM) was applied intracellularly to a third neuron without effect (lower trace). CABA was applied for the duration indicated by the solid line. (B) The stability plot for GABA-activated currents obtained from 7-12 neurons is plotted for cells exposed to control pipette solution, f500 nM PDBu, or +500 nM a-PDD. All points are means + SEM. Holding potential was -50 mV.

constants for decay when compared with the controi neurons not exposed to phorbcl esters.

Discussion

We have studied the functional effects and the sites of phosphorylation on GABAA receptor subunits by PKC using recombinant GABAh receptors and site- directed mutagenesis to ascertain the importance of the serine residues, 409 on 01 and 327 and 343 on y2 subunits, that are recognized as substrates for PKC- induced phosphorylation (Moss et a!., 1992a).

Biochemical Localization of the Sites of Phosphorylation Biochemical analyses of GABAn receptor phosphorylation were tised to analyze subunit-specific phosphorylation. Unfortunately the susceptibility of the

CABA* Receptors and PKC 1091

y2 subunit to proteolysis limited this approach to the al and PI subunits. S409 was the major site of phosphorylation in the l31 subunit, as determined by site- specific mutagenesis. Interestingly, this residue is also phosphorylated by PKA (Moss et al., 1992a, 1992b) and by calciumlcalmodulin-dependent protein kinase II and cGMP-dependent protein kinase (McDonald and Moss, unpublished data). Since this site (S409) is conserved in all b subunits isolated to date (Swope et al., 1992), these results suggest a crucial role for 0 subunits in mediating the effects of phosphorylation.

Phosphorylation Modulates GABAA Receptor Function Our studies with recombinant and native neuronal GABAA receptors have consistently revealed that a major feature of PKC phosphorylation induced by phorbol esters is a negative modulation of receptor function. However, phorbol esters were noted to cause small transient enhancements in the CABA-activated responses, but this effect was reproduced by the inactive a phorbols and was also observed with the B phorbols applied to mutated GABAA receptors devoid of PKC phosphorylation acceptor sites. Overall, these results suggest that this enhancement is probably not a feature associated with direct phosphorylation of the GABAA receptor by PKC. This is apparent from the reduced amplitudes of GABA-activated currents in A293 cells and Xenopus oocytes, in addition to the mixed/noncompetitive depression in the GABA concentration-conductance curves. The development of the depression in the GABA-induced currents has a timecourseconsistentwith second messenger media- tion, taking at least 5-10 min before any effect is apparent and for the injected Xenopus oocytes, up to 20- 30 min. These effects of the phorbol esters on the GABAA receptor subunits were abolished by mutating serine residues in PI, y2S, and y2L subunits identified from biochemical studiesas keysitesfor phosphorylation by PKC.

Recordings from A293 cells expressing GABAA receptors comprising alp’ly2S or alply2L subunits responded to GABA with a decaying current that was fitted by a single exponential. Phorbol esters did not apparently affect the time constant for decay; however, for receptors composed of alB1 subunits, a dou- ble exponential rate of decay was evident, with phorbol esters reducing the fast component of decay without affecting the underlying time constants. Thus phosphorylation per se seems to have little effect on the kinetics of GABA-activated membrane currents. This result with phorbol esters activating PKC was reminiscent of previous resultsobtained using similar recombinant receptors and PKA (Moss et al., 199213). In receptors comprising aIS subunits, the only site for phosphorylation by PKA and PKC is S409, so it is probably not surprising that these kinases have similar functional effects. The addition of the y2 subunit

to the GABAA receptor complex does confer up to 2 additional sites for phosphorylation by PKC that are not phosphorylated by PKA. Our studieswith alply2S and al(31y2L GABAA receptors in Xenopus oocytes suggested that phosphorylation at any of these sites, all studied separately,will producea negative modulation of receptor function. However, phosphorylation at S343, which is contained in the 8 extra amino acids inserted in the alternatively spliced long version of y2 (Whiting et al., 1990; Kofuji et al., 1991), seems to produce the largest effect, suggesting that the sites of phosphorylation are not functionally equivalent. It is possible that the conformation of the large intracellular loop between transmembrane domains 3 and 4 is altered by the insertion of the alternatively spliced region of amino acids and that subsequent phosphorylation has a more profound effect. It is notable that phosphorylation at S409 in the 81 subunit, in addition to phosphorylation at S327 and S343 in the y2L subunit, does not produce a further significant reduction in the GABA-activated current, suggesting that the functional effects of phosphorylation at the 3 serines do not sum linearly. Another intriguing difference between the sites of phosphorylation is the type of inhibition of the GABA concentration-response curves. Phosphorylation at either S327 or S343 in the y2 sub- unitcaused asmall lateral shift in thecurve in addition to a depression of the maximum GABA-induced conductance. This mixed inhibition was not seen with aIf wild-type GABAA receptors, or when both sites in the y2L subunit were mutated to alanines in alpI- ~2L(S327A,S343A). This shift could be interpreted as an effect of phosphorylation on agonist affinity and/ or steps leading up to ion channel activation.

The inhibition caused by PKC-induced phosphorylation of the (jl and y2 receptor subunits observed in this study is not due to the expression of recombinant GABAA receptors in an atypical membrane environ- ment, since similar data were obtained by studying native GABAA receptors in sympathetic neurons. It is conceivable that GABAA receptors expressed in some other neurons may respond differentlyto phosphorylation, but the recombinant receptor with composition alply subunits does reproduce much of the in situ pharmacology seen in many different neuronal preparations. Our attempts to express aly2S or aly2L GABAA receptors failed to produce functional chan- nelsineitherA293cellsorXenopusoocytes(seeAnge- lotti et al., 1993).

Comparison with Previous Results Previous studies of the effect of PKC-induced phosphorylation on GABAA receptor function have reported a variety of results. Sigel and colleagues observed an inhibition of GABA-activated currents in whole brain mRNA-injected Xenopus oocytes that was not reproduced by an inactive phorbol (Sigel and Baur, 1988; Moran and Dascal, 1989). A similar profile for the inhibition of responses to GABA is also evident

Neuron 1092

on recombinant GABAA receptors (Sigel et al., 1991). Interestingly, it was noted that responses to lower concentrations of GABA were more reduced by phosphorylation compared with a saturating CABA concentration. This analysis was performed with receptors comprising rat a5f32y2S and is entirely consistent with a mixed type of inhibition for phosphorylation, as reported in our study for YZcontaining receptors. However, for alPI receptors, a dependence of inhibition on the GABA concentration was not apparent. This is compatible with a noncompetitive mode of inhibition that was also noted by Leidenheimer et al. (1992) after exposing GABAA receptors to PMA in mouse brain mRNA-injected Xenopus oocytes. Sigel et al. (1991) reported that the inhibition of responses to GABA by phorbol esters was unaffected by the type of y2 subunit present, i.e., either y2S or y2L. In contrast, we have observed a small difference with receptors containing the y2L subunit, which accentuates the depression of GABA-activated responses by PKC- induced phosphorylation. This difference became evident only on constructing full concentration- response curves and was not readily apparent at lower concentrations of GABA producing responses of 5%- 40% of the maximum (see Sigel et al., 1991). The mutations of the 3 serine residues in 01 and y2 subunits blocked the functional effects of PKC-induced phosphorylation following activation by phorbol esters. Curiously, these mutations did not completely elimi- nate the inhibition by phorbol esters in a recent study by Kellenberger et al. (1992) using GABA* receptors comprisng alfi2y2S subunits. After exposure to PMA for only 1.5 min, responses mediated bywild-type GA- BAA receptors were more reduced compared with the mutant alg2(S410A)$YS(S327A) receptors, but after in- cubation for IO-15 min, both responses were equally reduced by PMA (Kellenberger et al., 1992). Our biochemical studies on the PI subunit demonstrated that mutation of S409 resulted in insignificant levels of phosphorylation. If this can be extrapolated to the ~2 subunit, we would expect the functional effect of PKC to be clearly affected by these mutations.

In comparison with studies on expressed GABAA receptors, PKC-induced phosphorylation of native neuronal GABAA receptors in mouse spinal neurons by PDBu did not affect GABA-induced 36CI-flux (Ticku and Mehta, 1990). In contrast, exposure of cortical or cerebellar microsacs to PMA reduced muscimol- activated 36CI-flux(Leidenheimer et al., 1992). Some of these different results might be ascribed to receptor heterogeneity or differences in the species of PKC and the efficacy of the kinase to phosphorylate GABAA receptor proteins; alternatively, a selective compartmentalization of PKC and CABAA receptors may exist in some neurons. In adult dissociated hippocampal neurons, GABA-activated currents are slowly inhibited over 25 min by 250 nM PDBu-an effect shared by the diacylglycerol analog I-oleoyl-2-acetylglycerol but not by a-phorbol 12,13-dibutyrate (Stelzer, 1992). These effects are similar to the action of PDBu on

GABA-activated currents recorded in cultured sympathetic neurons. Moreover, the phosphorylation of these neuronal GABA* receptors by PKC showed many similarities with that previously reported for PKA-induced phosphorylation (Moss et al., 1992b).

Physiologica! Significance of Phosphorylation Molecular and biochemical analyses have revealed that GABAA receptor subunits are substrates for a number of protein kinases (Swope et a!., 1992). A pre- cise invariant amino acid consensus sequence for phosphory!ation by PKC has not yet been reported. The most prevaient sequence involves 1-3 basic amino acids (R or K) located N- and C-terminal to the phosphoacceptor serine or threonines (S/T) and separated by up to 2 amino acids (X) regarded as recogni- tion neutral according to the consensus R/K1.3X2-OS/ TX2&/K,-3 (Kennelly and Krebs, 1991). Therefore, although it is possible that other consensus sequences for phosphorylation by PKC exist in the GABAA receptor subunit families, to date the main targets for PKC appear to be the B subunit family and $2 subunits (Swope et al., 1992; Raymond et ai., 1993).

Using heterologous expression, our results demonstrate that the functional consequences of PKC- induced phosphorylation of the GABAn receptor is dependent on the receptor subunitcomposition.This is highlighted in the case of the ~2 subunit, in which the addition of only8 amino acids, which incorporate a consensus site for PKC, can alter the degree of modulation by phosphorylation. It is interesting that phosphorylation of this “extra site” may also underly the ethanol potentiation of GABA-activated responses (Wafford et a!., 1991; Wafford and Whiting, 1992; although see Sigel et al., 1993). Overall, the regional variations in GABAA receptor structure might confer a different susceptibility to regulation by phosphorylation, which could be mediated by alternative signal transduction pathways. This could lead to a long-term modulation of receptor function. A fine tuning of the level of modulation might be achieved by selective compartmentalization of GABA,, receptors and protein kinases. Presently, it is not known whether ali types of PKA and PKC can phosphorylate GABAA receptors and cause functional changes. It will be of interest and physiological significance to determine whether PKC-induced phosphorylation of GABAA receptors will affect synaptic inhibition mediated via GABA,, receptors, as has been proposed for phosphorylation via PKA (Soldo et al., 1991).

Experimental Procedures

Expression Vectors The cloning of the murine al, yZS, and y2L cDNAs has been previously described (Kofuji et al., 1991; Wang et al., 1992). A 81 CABAA receptor cDNA was isoiated from a mouse brain cDNA library by low stringency hybridization. The murine Bl subunit cDNA has 2 sequence nearly identical to that of the rat (Ymer etal., 1989),with theexceptionofasingleaminoacid substitution at position 322, where a lysine residue (rat) is changed to an arginine (mouse). These cDNAs were cloned as EcoRi fragments

GABAA Receptors and PKC 1093

into the mammalian expression vector pGW1 (Moss et al., 1991). Site-specific mutagenesiswas performed as described by Kunkel et al. (1985) using the following primers to make substitutions as described previously (Moss et al., 199213): S409 to alanine @I subunit), TFTCAGCTCCGCCCCGCCCCT; S343 to alanine (y2L subunit), GCCCTTGAAGCCAAACAT; and S327 to alanine (y2S and y2L subunits), TTTATCCTTGCCTGGCTf. The fidelity of the final expression constructs was verified by DNA sequencing. The PKCa expression construct SRa-PKCa (Kaibuchi et al., 1989) was a generous gift from Dr. K. Kaibuchi (DNAX Research In- stitute).

Antibody Production A glutathione S transferase fusion protein encoding the large intracellular domain of the murine CABAA receptor Bl subunit (Moss et al., 1992a) was used to immunize New Zealand White rabbits (Hazelton). Antisera were affinity purified on a column prepared by coupling the fusion protein to Affi-Gel 15 (Bio-Rad). Antiserum to the murine CABAA receptor al subunit was raised against a synthetic peptide, KQPSQDELKDNTTVFTRILDR, corresponding to the N-terminus of this subunit, which was synthe- sized on an Applied Biosystems model 380A peptide synthesizer. The peptide was coupled with glutaraldehyde (Sigma grade 1) to thyroglobulin or bovine serum albumin (Harlow and Lane, 1988). Antiserum against the thyroglobulin-conjugated peptide was raised in New Zealand White rabbits (Hazelton) by standard procedures (Harlow and Lane, 1988). The antiserum was affinity purified on a column prepared by coupling the bovine serum albumin-conjugated peptide to Affi-Gel 15.

Cell Preparation Human Embryonic Kidney Cells Human embryonic kidney cells (ATCC CRL1573) were grown in Dulbecco’s modified Eagle’s medium and Ham’s F12 supplemented with 10% fetal calf serum at 37OC in 95% air, 5% CO, (Moss et al., 1991; Smart et al., 1991). Cells were fed weekly and subcultured. Exponentially growing cells were used for transfection, seeded onto 35 mm plastic dishes, and viewed with a Nikon Diaphot microscope for electrophysiology. cDNAs corresponding to murine GABAA receptor subunits al, Bl,y2S, and y2Lwere incorporated intoA293cells usingamodifiedcalcium phosphate transfection technique (Chen and Okayama, 1987). A293 cells were used for recording 18 to 72 hr after transfection and had membrane potentials ranging from -30 to -60 mV. Superior Cervical Ganglion Neurons Cultured embryonic rat sympatheticganglion neuronswerepre- pared as described previously (Smart, 1992). Briefly, gangliawere removed from embryonic day21 ratsand pooled prior todissoci- ation using enzymatic and mechanical trituration. Dissociated neurons were grown on a laminin substratum in Leibovitz’s medium supplemented with 10% (v/v) fetal calf serum, 2 mM gluta- mine, 0.6% (w/v) glucose, 0.19% (w/v) NaHCO,, 100 U/ml penicil- lin-G, 100 pglml streptomycin, and 50 nglml 7S-nerve growth factor. Neurons were incubated at 37OC in 95% air, 5% CO*. Cells were fed with fresh medium twice weekly after controlling Schwann cell and fibroblast growth with 10 PM cytosine arabino- side for 24 hr. Neurons were used after 3-14 days in vitro and possessed membrane potentials of -50 to -70 mV and action potential amplitudes of 80 to 120 mV. Xenopus Oocytes Oocytes were extracted from the ovaries of anesthetized Xeno- pus and placed in a modified Barth’s medium (MBM) comprising 110 mM NaCI, 1 mM KCI, 2.4 mM NaHCO,, 7.5 mM Tris-HCI, 0.33 mM Ca(NO&, 0.41 mM CaCb, 0.82 mM MgS04, and 50 pgl ml gentamycin (pH 7.6). Oocytes at stages V and VI were injected into the nucleus with 15-20 nl of 0.33-0.5 mglml DNA (encoding the murine GABAA receptor subunits al, Bl, y2S, and y2L). In- jected oocytes were incubated at 19OC for 2-3 days in MBM to permit the expression of receptor proteins and fed with fresh MBM every 2-3 days. Oocytes were subsequently stored at 1OoC in MBM to extend their survival time to 3-4 weeks. Cells possessed membrane potentials of -30 to -60 mV and input resistances of 1 to 5 Ma.

Cell Transfections, lmmunoprecipitations, and Phosphopeptide Mapping A293cells were transfected with GABAA receptor expression constructs with or without a cDNA encoding PKCa in equimolar ratios (20 pg total DNA) using calcium phosphate coprecipitation asdescribed previously(Mossetal.,1992a,1992b). In someexper- iments a cDNA encoding B-galactosidase was included. The transfection efficiencies, assessed by B-galactosidase staining of a proportion of the cells transfected with GABA* receptor constructs, varied by up to 30% * 20% between different dishes of cells during a single experiment. After 48 hr following transfection, cells were labeled with [3SS]methionine Translabel (0.25 mCi/ml; ICN/Flow) in methionine-free medium for 4 hr, or with [32P]orthophosphoric acid (0.5 mCi/ml; Amersham) for 4 hr. PKC was stimulated by the addition of 50 nM PMA for 15 min. GABAA receptors were immunoprecipitated under native conditions as described previously (Moss et al., 1992b). For denaturing immu- noprecipitations, cells were lysed by mild sonication in a buffer containing 20 mM sodium phosphate (pH 7.0), 50 mM sodium fluoride, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 150 mM NaCI, 5 mM EGTA, 5 mM EDTA, 1 mM phenylmethylsul- fonyl fluoride, 1 mM Benzamidine, 10 pg/ml leupeptin, 10 Kg/ ml antipain, 10 pglml pepstatin, and 10 U/ml aprotinin. A crude membrane preparation was prepared by centrifugation (100,ooO x g), and the membranes were solubilized in the above buffer supplemented with 2% Triton and 0.5% deoxycholate. GABAA receptor subunits were then isolated using affinity-purified antisera against the al and Bl subunits coupled to protein A-Sepha- rose. Receptor complexes were finally resolved by SDS-polyacrylamide gel electrophoresis using 9% gels. Bands were visualized by autoradiography. The level of phosphorylation was quantified by excision of the relevant bands followed bycheren- kov counting. Phosphopeptide mapping and phosphoamino acid analysis on excised gel slices were performed as previously described by Miles et al. (1989).

Electrophysiology Whole-Cell Recording ExperimentsonA293cellsandcultured neuronswereperformed using a List EPC7 amplifier in the whole-cell recording mode. Patch electrodes (I-5 MD) were fabricated from thin-walled borosilicate glass and filled with a solution containing 148 mM KCI, 2 mM MgClz, 1 mM CaCI,, 10 mM HEPES, 11 mM ECTA, and 2 mM ATP. The cells were viewed under phase-contrast optics and continuously superfused with a Krebs’ solution containing 140 mM NaCI, 4.7 mM KCI, 1.2 mM MgClz, 2.5 mM CaC&, 10 mM HEPES, and 11 mM glucose. Series resistance compensation of 80% was routinely achieved. Membrane currents were filtered at 10 kHz (-3 dB, 6 pole Bessel filter, 36 dB/octave) and recorded on a Racal store 4D FM tape recorder (DC to 5 kHz) and a Brush- Gould 2200 ink-jet pen recorder. Intracellular recording Membrane currents were recorded from Xenopus oocytes, re- taining their follicular cell envelope, using a two electrode voltage-clamp method. Oocytes were superfused at 8-10 mllmin (bath volume, 0.5 ml) with an amphibian Ringer’s solution containing 110 mM NaCI, 2 mM KCI, 5 mM HEPES, and 1.8 mM CaCb (pH 7.4). Both the microelectrodes were fabricated from thin-walled borosilicate glass and filled with 3 M KCI (voltage) and 0.6 M K2S04 (current), providing resistances of 5-10 MD and l-2 MD, respectively. An Axoclamp-PA amplifier was used in voltage-clamp mode, and all data were recorded on a Gould 2200 ink-jet pen recorder. Analysis of Membrane Conductance The GABA-induced membrane conductance was calculated by subtraction from the resting membrane conductance. Conduc- tances were ascertained by briefly stepping the membrane potential from a holding potential range of -30 to -50 mV with a small amplitude voltage command step (1 s duration, -10 mV amplitude, 0.2 Hz frequency) in the absence and presence of GABA.Thesedatawereused toconstruct equilibriumconcentra- tion-response relationships for CABA. The data were fitted with a logistic state function of the form

NWKJll 1094

GIG,,, = l/j1 + WG4W~, where G and Cm,, represent the GABA-induced conductance at a grven concentration and the maximum conductance induced by a saturating concentration of GABA, respectively. EC,, defines the concentration of GABA ([A]) that induces a half-maximum response, and n is the Hill coefficient.

Drugs and Solutions

Phorbol esters were dissolved in ethanol to a final stock concentration of 1 mM. Dilutions for application to our cells were made with Krebs’ or Ringer’s solution, and the final concentration of ethanol did not exceed 0.03% (v/v). At this concentration of ethanol, all control recordings of GABA-activated responses in A293 cells, Xenopus oocytes, or cultured neurons were unaffected by the solvents. To obtain the fast kinetic data, drugs were applied close to (50-100 urn) neurons or transfected AZ93 cells using a multibarreled electrode fabricated from Quad glass tubing (XGC300-10, Clarks Electromedical, England). The operative bar- rel was switched using solenoid valves, and solution exchange was effected in these experiments within S-IO ms.

Acknowledgments

This work was supported by the Howard Hughes Medical Insti- tute, the Medical Research Council (UK), and the Wellcome Trust. We are grateful to Martin Raff and Ann Mudge (University College, London) for additional support. 5. J. K. and X. X. contrib- uted equally to this study.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received January 19, 1994; revised March 8, 1994.

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GABA* Receptors and PKC 1095

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Note Added in Proof

The work cited as McDonald and Moss, unpublished data, is now in press: McDonald, B. J., and Moss, S. J. (1994). Phosphoryla- tion of the intracellular domains of y-aminobutyric acid type A receptor subunits by calciumlcalmodulin type II dependent protein kinase and cyclic GMP dependent kinase. J. Biol. Chem., in press.

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Wang, J. B., Kofuji, P., Fernando, J. C., Moss, S. J., Huganir, R. L., and Burt, D. R. (1992). The alpha 1, alpha 2, and alpha 3 subunits of GABAA receptors: comparison in seizure-prone and -resistant mice and during development. J. Mol. Neurosci. 3, 177-184.

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Regulation of GABAA receptor function by protein kinase C phosphorylation