Japanese Journal of Physiology, 50, 429–435, 2000

Activation of Protein Kinase C Induces Internalization of the GABAC Receptors Expressed in Xenopus Oocytes

Tadashi KUSAMA, Kouji HATAMA, Kiyoshige SAITO, Yasuo KIZAWA, and Hajime MURAKAMI

Department of Physiology and Anatomy, Nihon University College of Pharmacy, Funabashi, 274–8555 Japan

Abstract: In a previous study, we showed that change in affinity for GABA. To allow histochemi- the protein kinase C (PKC) activator 4␤-phorbol cal detection of ␳1 GABAC receptors, we con- 12-myristate 13-acetate (PMA) inhibited the ␥- structed a receptor tagged at the C-terminal po- aminobutyric acid (GABA)Ðgated currents in sition with human c-myc epitope. Electrophysio- Xenopus oocytes expressing human ␳1 GABAC logically, the tagged receptors showed almost the receptors. To investigate whether the inhibition of same sensitivities for GABA and PMA as those currents was due to a decrease in efficacy or in of wild-type ␳1 GABAC receptors. Immunohisto- the potency of ␳1 GABAC receptor, concentra- chemistry with anti-myc antibody detected a tion-response curves for GABA were compared dense concentration of tagged receptors at the before and after PMA treatment. The EC50 con- surface area of Xenopus oocytes. Transient ex- centrations of GABA obtained during the maxi- posure to PMA reduced the density of immuno- mally inhibited period were not statistically differ- fluorescence at the surface area and increased it ent from the concentrations obtained before PMA in the subsurface area. These results suggest treatment (1.74Ϯ0.33 and 1.45Ϯ0.28 ␮M, re- that the stimulation of protein kinase C leads to spectively). These results indicate that the inhibi- internalization of ␳1 GABAC receptors expressed tion depends on a change in number or conduc- in Xenopus oocytes. [Japanese Journal of tance of active receptor channels, but not on a Physiology, 50, 429Ð435, 2000]

Key words: GABAC receptor, protein kinase C, Xenopus oocyte, internalization, immunohistochem- istry.

␥-Aminobutyric acid (GABA) is a major inhibitory channels and are thought to mediate inhibitory infor- neurotransmitter in the mammalian central nervous mation, they have distinct pharmacological and bio- system, and its action is mediated by three types of physical properties. GABAC receptors are not sensi- GABA receptors. The GABAA receptor, the predomi- tive to GABAA receptor modulators (barbiturates, nant type, is a ligand-gated ion channel composed of benzodiazepines, and neurosteroids), antagonists several subunits [1, 2] and contains modulatory sites (bicuculine) or GABAB receptor agonists [11–13]. In for benzodiazepines, barbiturates, neurosteroids, and particular, GABAC receptors show high affinity for ethanol [2–4]. The GABAB receptor, the second type, GABA and muscimol, and are specifically activated is a member of the seven transmembrane family [5] by a GABA analogue cis-(2-(aminomethyl)-cyclo- and is linked to Kϩ or Ca2ϩ channels via G-proteins. propylcarboxylic acid [14]. A specific competitive in- The GABAC receptor, the third type, has been de- hibitor of GABAC receptors, (1,2,5,6-tetrahydropyri- scribed in literature to be highly expressed in the dine-4-yl)methylphosphinic acid (TPMPA), has also retina of several species [6–10]. Although the GABAA been described [15]. These characteristics of GABAC and GABAC receptors are both ligand-gated chloride receptors are faithfully reproduced in heterologous ex-

Receive on February 10, 2000; accepted on June 21, 2000 Correspondence should be addressed to: Tadashi Kusama, Department of Physiology and Anatomy, Nihon University College of Phar- macy, 7–7–1, Narashinodai, Funabashi, 274–8555 Japan. Tel: ϩ81–47–465–4789, Fax: ϩ81–47–465–4789, E-mail: [email protected] u.ac.jp

Japanese Journal of Physiology Vol. 50, No. 4, 2000 429 T. KUSAMA et al.

pression studies of GABA receptor ␳-subunits cloned (Drummond). Oocytes were incubated for 2 to 7 d at from the retina of a number of species [16–21]. In ad- 19°C in ND96 medium containing 1.8 mM CaCl2, Ϫ Ϫ dition to these differences, recent molecular evidence 2.5 mM pyruvate, 50 U ml 1 penicillin and 50 ␮gml 1 strengthens the classification of ␳-subunits in a sepa- streptomycin. rate receptor group: ␳-subunits do not combine [22] Electrophysiological experiments and data nor co-localize [23] with GABAA receptor subunits. analysis. The oocyte was placed in a 0.5-ml bath Recently, MAP-1B, a linker protein that specifically chamber, which was perfused continuously Ϫ1 links GABAC but not GABAA receptors to the cy- (2 ml min ) with ND96 with 1.8 mM CaCl2 at 19°C, toskeleton, was discovered [24]. Although the phar- and voltage clamped using a CEZ-1250 amplifier macology and molecular biology of GABAC receptors (Nihon Kohden, Japan) at a holding potential of have been studied in detail, very little is known about Ϫ70 mV with two glass capillary microelectrodes the mechanisms of modulation in their activities as re- filled with 3 M KCl. A near-maximal concentration of ␮ ceptor channels except that ␳1 GABAC receptors ex- GABA (10 M) was applied every 15 min. Peak cur- pressed endogenously in retina are down-regulated by rent amplitude was used for data calculation for all ex- protein kinase C (PKC) [25]. periments. After the amplitude of GABA currents be- In a previous study, we showed that the PKC activa- came constant, 25 nM PMA (Sigma) was applied for tor 4␤-phorbol 12-myristate 13-acetate (PMA) inhib- 3 min by superfusion. Currents are standardized to the ited the GABA-gated currents in Xenopus oocytes ex- control currents just prior to the application of PMA. pressing human ␳1 GABAC receptors [26]. The pres- For GABA concentration–response curves, responses ent study was undertaken to obtain a molecular insight to GABA (0.1–100 ␮M) were assessed using control into the mechanism underlying the inhibitory effect of (untreated) oocytes and the same responses were as- PKC on ␳1 GABAC receptors. In order to visualize sessed again 60 to 120 min after the PMA treatment the receptors expressed in Xenopus oocytes, we con- using the same oocyte. Concentration-response data is structed c-myc epitope–tagged ␳1 GABAC receptors. normalized as the percentage of the maximum GABA Immunohistochemistry using anti-myc antibody response obtained in the control with GABA 100 ␮M showed the localization of tagged receptors at the sur- being taken as the maximum. Higher concentrations face area of Xenopus oocytes and their redistribution of GABA did not elicit larger currents in the control by PMA treatment. or PMA-treated oocytes. EC50 values were calculated using SigmaPlot (Jandel Scientific, Corte Madera, Ϯ MATERIALS AND METHODS CA). Results are expressed as means SEM. Statisti- cal analysis was performed by Student’s t-tests where DNA constructs and RNA synthesis. The appropriate. PMA was dissolved in dimethyl sulfoxide ␮ human ␳1 GABAC receptor [16] was inserted into the (DMSO) at a concentration of 25 M and diluted in mammalian expression vector pcDNA I (Invitrogen) ND96 with 1.8 mM CaCl2 just before use. by standard methods to produce pcDNA1-␳1. The Immunohistochemical studies. Immunoflu- human c-myc epitope GEQKLISEEDL [27] was intro- orescence detection in oocytes was performed using duced into the carboxyl terminus of the ␳1 GABAC re- slices of fixed oocytes. Oocytes were injected with ceptor by polymerase chain reaction (PCR). The PCR 10 ng of c-myc–tagged ␳1 GABAC receptors and product was subcloned into pcDNA I/Amp (Invitro- processed for immunofluorescence after 4 d of expres- gen). Capped cRNA for ␳1 GABAC receptor and c- sion. We electrophysiologically selected oocytes myc epitope–tagged ␳1 GABAC receptor were synthe- showing nearly the same amplitudes of GABA cur- sized in vitro from the linearized plasmids by using a rents and separated them into two groups. One group T7 mMESSAGE mMACHINE kit (Ambion). was incubated in 25 nM PMA for 3 min. The other was Xenopus oocyte expression. Adult female used as the control without PMA. At 60 min after the Xenopus laevis were anaesthetized with ice. The last rinse with ND96 with 1.8 mM CaCl2, oocytes oocytes were removed and treated with 2 mg mlϪ1 col- were incubated in a 30% sucrose ND96 medium for lagenase (Sigma, Type IA-S) in Ca2ϩ-free ND96 solu- 30 min. Then the oocytes were fixed for 3 h in 0.01 M tion (composition, mM: NaCl 96, KCl 2, MgCl2 1, N- phosphate-buffered saline (PBS) solution containing (2-hydroxyethyl)-piperazine-NЈ-(2-ethanesulphonic 4% formaldehyde. Fixed oocytes were embedded in acid) (HEPES) 5, pH 7.5) as described previously Tissue-tek embedding medium (Sankyo), frozen, and [14]. Stage 5 and 6 oocytes were manually defollicu- sliced (14 ␮m thickness) on a CR-502 cryostat (Yam- lated and injected with 5 to 10 ng of capped cRNAs in ato). After blocking with 20% goat serum in PBS for 10 nl of water using a positive-displacement pipette 1 h, slices were incubated with anti-myc rabbit anti-

430 Japanese Journal of Physiology Vol. 50, No. 4, 2000 Internalization of GABAC Receptor by PKC

body (Santa Cruz Biotech, 2 ␮g/ml) in a wet chamber ceptors in Xenopus oocytes by a histochemical at 4°C for 12 to 16 h. The slices were then incubated method. The sensitivities of the tagged receptors to for 2 h with secondary antibody (anti-rabbit IgG cou- GABA and PMA were compared with those of the pled to fluorescein isothiocyanate (FITC), 1 : 300; wild-type ␳1 GABAC receptors. The tagged receptors Biomedical Tech). After each step, the sections were evoked nearly the same current amplitude as the wild- rinsed and washed in PBS containing 0.3% Tween-20 type receptors in response to GABA (1,150Ϯ32 and (2ϫ30 min). 1,168Ϯ27 nA for 100 ␮M GABA and 1,055Ϯ42 and 1,072Ϯ35 nA for 10 ␮M GABA, respectively). Treat- RESULTS ment with 25 nM PMA for 3 min inhibited the currents through the tagged receptors in a time-dependent PMA does not reduce receptor affinity for manner. Although the onset of inhibition of tagged re- GABA ceptors was somewhat delayed, the plateau levels of Previously, we have found that transient treatment inhibition at 60 min after PMA were not different with PMA inhibits the GABA-gated currents through from those of the wild-type ␳1 GABAC receptors (Fig. the human ␳1 GABAC receptors expressed in Xenopus 3). oocytes [25]. This time, we investigated the duration of inhibition and compared the affinity for GABA be- fore and after PMA treatment. The inhibition devel- oped rapidly, reaching a maximum about 40 min after the application of 25 nM PMA for 3 min (Fig. 1). The inhibition remained maximal for at least 120 min and then subsequently recovered gradually, although re- covery was not complete even 24 h after treatment. Then we compared the concentration-response curves for GABA obtained before PMA treatment with those obtained 60 to 120 min after treatment in the same oocytes (Fig. 2A). The EC50 concentrations of GABA before and after treatment were not statistically differ- ent (1.45Ϯ0.28 and 1.74Ϯ0.33 ␮M, respectively), al- though the maximum responses induced by 100 ␮M Fig. 1. Time course of the PMA-mediated inhibition of GABA were inhibited from 1,168Ϯ27 to 203Ϯ9nA GABA-induced currents in Xenopus oocytes expressing ␳ by PMA treatment (Fig. 2B). These results suggest 1 GABAC receptors. Closed circles represent the mean ϭ ␮ that PMA inhibit ␳1 GABA receptors by reducing (n 3 to 6) amplitudes induced by 10 M GABA applied at C 15 min intervals plotted against the time before and after the number or conductive active receptor channels ex- 25 nM PMA, which was administered for 3 min as indicated pressed in the oocytes. by the dotted column at time 0. Each amplitude is shown We constructed an epitope-tagged ␳1 GABAC re- relative to the one just before PMA treatment. Vertical bars ceptor in order to visualize the expression of the re- show standard errors of the mean.

Fig. 2. Change in the GABA-induced cur- rents by treatment with PMA. A: Concentra- tion-response curves for GABA-induced cur- rents obtained before and after treatment with PMA. Open circles represent the mean (nϭ13) amplitudes for various concentrations of GABA as percentages of the maximum re- sponse induced by 100 ␮M of GABA in the oocytes before treatment with PMA. Closed circles represent the mean (nϭ4) amplitudes obtained 60 to 120 min after treatment ex- pressed as the percentage of the maximum GABA response obtained in the pre-treated oocyte. Vertical bars show standard errors of

the mean. B: Values plotted at A and EC50 val- ues obtained from A. Values are expressed as meansϮSEM.

Japanese Journal of Physiology Vol. 50, No. 4, 2000 431 T. KUSAMA et al.

Demonstration of internalization by immuno- histochemistry Oocytes showing similar amplitudes of GABA cur- rents (difference in maximal currents was within 10%) were selected electrophysiologically from those in- jected with 10 ng of cRNA for c-myc–tagged ␳1 GABAC receptor and separated into two equal por- tions. One portion was incubated with 25 nM PMA for 3 min and the other portion was treated with the vehi- cle. At 60 min after the PMA or vehicle treatment, oocytes were fixed and treated with antibodies for im- munohistochemistry. Figure 4 shows the in situ detec- tion of c-myc–tagged ␳1 GABAC receptors on 14 ␮m sections of oocytes. Fluorescence was detected only in the slices treated with primary anti-myc rabbit anti- body and secondary FITC-coupled anti–rabbit IgG antibody. The fluorescence signal was largely local- ized to the plasma membrane region, though some in- Fig. 3. Time course of PMA-mediated inhibition of ␳ tracellular labeling could also be observed (Fig. 4A). GABA-induced currents through c-mycÐtagged 1 GABAC receptors expressed in Xenopus oocytes. Exper- As expected, no such specific fluorescence could be imental conditions were the same as in Fig. 1. Closed cir- detected when a single antibody was used for detec- cles represent the mean (nϭ4) amplitudes of oocytes ex-

tion or when oocytes expressing the wild-type ␳1 pressing c-myc–tagged ␳1 GABAC receptors induced by ␮ GABAC receptors were treated with both of the anti- 10 M GABA applied at 15 min intervals plotted against the bodies (data not shown). In the PMA-treated oocytes, time before and after 25 nM PMA, which was administered surface fluorescence was pale but dense linear fluores- for 3 min as indicated by the dotted column at time 0. Open circles represent the amplitudes of oocytes (nϭ13) ex- cence drawn back from the surface to the inner area pressing the wild-type ␳1 GABAC receptors for reference. was observed (Fig. 4B). Such patterns of fluorescence Each response is shown relative to the response just before indicating redistribution of the tagged receptors by PMA treatment. Vertical bars show standard errors of the PMA were also obtained in the sections of oocytes mean. from three different animals examined. AB

Fig. 4. Immunohistochemistry detects the internaliza- batch and had previously been tested electrophysiologically tion of c-mycÐtagged ␳1 GABAC receptors expressed in for similar expression of tagged ␳1 GABAC receptors. Xenopus oocytes after PMA treatment. Cryosections of Oocytes were also treated at the same time with the sec- paraformaldehyde-fixed oocytes were assayed with primary ondary antibody (FITC-coupled anti–rabbit IgG). A: Control rabbit antibody directed against human c-myc epitope, (vehicle treated). B: PMA treated. Scale bar for A and B, which was conjugated to the C-terminal position of ␳1 25 ␮m. Upper left from the bundle of fluorescence indicates GABAC receptors. Oocytes were derived from the same inner side of the oocyte for both panels. 432 Japanese Journal of Physiology Vol. 50, No. 4, 2000 Internalization of GABAC Receptor by PKC

DISCUSSION tagged receptors, the maximal levels of inhibition were not different from those of the wild-type re- Previously, we observed that PMA, a potent activator ceptors. These results suggested that the C- of PKC, inhibited currents through the human ␳1 terminus–tagged ␳1 GABAC receptors exhibited the GABAC receptors expressed in Xenopus oocytes [26]. same sensitivity to GABA and PMA as the wild-type The inhibition could be the result of changes in the ␳1 receptors. We then performed immunohistochem- GABAC receptor protein (e.g., direct post-transla- istry using this tagged receptor. Cryosections of tional modification by, for example, phosphorylation) paraformaldehyde-fixed oocytes, previously injected or interaction with some modifying factor, leading to and shown to display similar amplitudes of GABA altered function or inactivation. Alternatively, mem- currents, were assayed with primary rabbit antiserum brane trafficking of the ␳1 GABAC receptors could be directed against the human c-myc epitope and FITC- altered, leading to a reduction in the total number of coupled secondary antiserum directed against rabbit receptor protein molecules in the membrane. As a first IgG. Bright fluorescence appeared at the surface area approach to studying the PKC-induced inhibition of of the oocytes (Fig. 4A). The fluorescence was not ␳1 GABAC receptors, we removed all putative consen- caused by non-specific attachment of the secondary sus sites in the intracellular loops for potential PKC- antibody, because it was not observed when the pri- mediated phosphorylation. However, all of the mu- mary antibody was omitted (data not shown). PMA tants were still as sensitive to PMA as the wild-type treatment (25 nM, 3 min) showed a clear redistribution receptor [28]. These findings suggested that non- of receptor staining from the surface to the inner area, canonical PKC phosphorylation sites might exist or with a linear configuration (Fig. 4B). As the oocytes the phosphorylation of consensus sites by PKC is not in Fig. 4 were derived from the same batch, displayed responsible for the inhibition of ␳1 GABAC receptors nearly the same amplitudes of GABA-induced current by PMA. Alternatively, PKC activation could lead to and treated at the same time for histochemical assay, the phosphorylation of regulatory proteins associated the change in fluorescence indicates the change in dis- with membrane trafficking of the ␳1 GABAC receptor tribution of ␳1 GABAC receptors by PMA. Two possi- protein. bilities exist for giving such a redistribution pattern; In this study, we further tried to address the mecha- one is simple infolding of the membrane expressing nism of inhibition, which could be due to (1) a de- tagged GABAC receptors and the other is the internal- crease in the affinity for GABA or (2) a decrease in ization of it. There are a couple of clues to settle this the number or conductance of functional receptor question. First, is the decrease in current through channels. Initially, we compared the affinities of the GABAC receptors expressed in Xenopus oocytes. In ␳1 GABAC receptors for GABA (Fig. 2). The EC50 the case of simple infolding, the total current would concentrations of GABA before and after PMA treat- not decrease because it is still possible for the outside ment were not statistically different (1.45Ϯ0.28 and of the folded membrane expressing the GABAC recep- 1.74Ϯ0.33 ␮M, respectively), although the maximum tors to be in contact with the extracellular medium responses induced by 100 ␮M GABA were inhibited containing GABA. On the other hand, in the case of from 1,168Ϯ27 to 203Ϯ9 nA. These results suggest internalization, the total current should be reduced, as that PMA inhibits currents through the GABAC recep- in our results, because the internalized membrane can- tors by reducing the number or the conductance of ac- not contact the extracellular GABA. Second, is the tive receptor channels expressed in the oocytes. To change of membrane capacitance (Cm) of the oocytes. study the former possibility, we constructed a human In the case of simple infolding, Cm measured electro- c-myc epitope–tagged GABAC receptor in order to be physiologically should increase because the surface able to visualize receptors in the Xenopus oocytes by membrane must extend to cave without a decrease in using histochemistry. Before the histochemical stud- the size of the oocyte. We did not find any decrease in ies, we confirmed by electrophysiological methods the size of the oocytes after treatment with PMA (data that the tagged receptor had the same sensitivity to not shown). In the case of internalization, on the other GABA and PMA as the wild-type receptor. The hand, two types of change are possible. To internalize tagged receptors evoked nearly the same current am- the surface membrane without changeing the size of plitude as wild-type receptors in response to 10 ␮M the oocyte, only the infolded portion must be internal- GABA (1,055Ϯ42 and 1,072Ϯ35 nA respectively). ized or the same volume of membrane as internalized The effects of PMA were compared between tagged must be supplied by a process similar to exocytosis. In receptors and wild-type receptors (Fig. 3). Although the former case, Cm will decrease, but it will not the onsets of inhibition were somewhat delayed at the change in the latter. Some laboratories have shown

Japanese Journal of Physiology Vol. 50, No. 4, 2000 433 T. KUSAMA et al. that exposure of oocytes to PKC activators causes a for these linker proteins, then GABAA and GABAC re- reduction in the Cm of oocyte indicating a reduction of ceptors should be differentially regulated by the acti- surface membrane [29–31]. This result, decrease in vation of PKC in mammalian neurons or retina. En- Cm, clearly denies the possibility of infolding by PMA dogenous ␳1 GABAC receptors are known to be treatment. Time-courses of reduction in Cm almost down-regulated physiologically by PKC [25], and here concurred with our time-course of reduction in GABA we showed the internalization of ␳1 GABAC receptors current by PMA. Third, is the real change in the struc- expressed in Xenopus oocytes by PKC. However, it re- ture of the surface membrane obtained by electron mi- mains to be clarified whether ␳1 GABAC receptors are croscopy. Vasilets et al. showed that PMA reduces the physiologically internalized by PKC in the retina and number and length of microvilli of Xenopus oocytes, differentially regulated from GABAA receptors. leading finally to a smooth surface membrane with a reduced surface area as shown by electron microgra- This study was supported, in part, by the Nihon University phy [29]. These findings all indicate that the redistrib- Research Grant for 1999. ution of immunofluorescence by PMA reflects an in- REFERENCES ternalization of the membrane expressing tagged re- ceptors rather than a simple infolding of it. Concern- 1. Davies P, Hanna MC, Hales TG, and Kirkness EF: In- ing the mechanism of decrease in the currents through sensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit. Nature 385: 820–823, 1997 GABAC receptors, a possibility of decrease in the con- 2. Macdonald RL and Olsen RW: GABAA receptor chan- ductance of receptor channels by PMA cannot be de- nels. Annu Rev Neurosci 17: 569–602, 1994 nied. However, it seems that most of the reduction in 3. Bormann J: Electrophysiology of GABAA and GABAB current is dependent on the internalization from sub- receptor subtypes. Trends Neurosci 11: 112–116, 1988 sequent consideration. The intensity of fluorescence 4. Doble A and Martin IL: Multiple benzodiazepine recep- tors: no reason for anxiety. Trends Pharmacol Sci 13: located at the outer part of the bundle reflects the den- 76–81, 1992 sity of actually functioning receptor channels. Thus, a 5. Kaupmann K, Huggel K, Heid J, Flor PJ, Bischoff S, striking decrease of fluorescence at that part clearly Mickel SJ, McMaster G, Angst C, Bittiger H, Froestl W, explains the decrease in the amplitude of GABA- and Bettler B: Expression cloning of GABAB receptors gated currents after PMA treatment, even though the uncovers similarity to metabotropic glutamate recep- histochemical data could make only semi-quantitative tors. Nature 386: 239–246, 1997 6. Feigenspan A, Wassle H, and Bormann J: Pharmacol- estimates. ogy of GABA receptor ClϪ channels in rat retinal bipo- Recently, Filippova et al. suggested the internaliza- lar cells. Nature 361: 159–161, 1993 tion of the human ␳1 GABAC receptors by PMA treat- 7. Lukasiewicz PD, Maple BR, and Werblin FS: A novel ment of HEK and COS cells [32]. However, their as- GABA receptor on bipolar cell terminals in the tiger sumptions were all derived from electrophysiological salamander retina. J Neurosci 14: 1202–1212, 1994 8. Lukasiewicz PD and Wong RO: GABA receptors on findings. Here, we show for the first time histochemi- C ␳ ferret retinal bipolar cells: a diversity of subtypes in cal evidence that human 1 GABAC receptors are in- mammals? Vis Neurosci 14: 989–994, 1997 ternalized by the activation of PKC in Xenopus 9. Qian H and Dowling JE: A novel GABA response from oocytes. Similar histochemical evidence indicating in- rod-driven horizontal cells of the white perch retina. ternalization by PMA, occurring without phosphory- Nature 361: 162–164, 1993 lation of consensus sites for PKC in the receptor it- 10. Qian H and Dowling JE: GABAA and GABAC receptors on hybrid bass retinal bipolar cells. J Neurophysiol 74: self, has also been reported for GABAA receptors 1920–1927, 1995 expressed in Xenopus oocytes [33, 34]. Thus, PKC 11. Shimada S, Cutting GR, and Uhl GR: GABA A or C re- may similarly regulate the function of both GABAA ceptor? GABA rho1 receptor RNA induces bicuculline, and GABAC receptors, although many differences barbiturate and benzodiazepine–insensitive GABA re- in pharmacological and physiological properties sponses in Xenopus oocytes. Mol Pharmacol 41: 683–687, 1992 have been shown for these receptors. However, two 12. Lukasiewicz PD: GABAC receptors in the vertebrate linker proteins, GABAA-receptor–associated protein retina. Mol Neurobiol 12: 181–194, 1996

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