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Expression of Mammalian Y-Aminobutyric Acid Receptors With Proc. Nati. Acad. Sci. USA Vol. 88, pp. 4318-4322, May 1991 Neurobiology Expression of mammalian y-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes (retina/membrane current/bicuculline sensitvity/barbiturates/picrotoxin) L. POLENZANI*, R. M. WOODWARDt, AND R. MILEDI Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717 Contributed by R. Miledi, January 28, 1991 ABSTRACT y-Aminobutyric acid (GABA), the major in- poly(A)+ RNA isolated from bovine retina, showed that hibitory neurotransmitter in mammalian brain, is known to retina RNA primarily expressed receptors to excitatory interact with two classes of GABA receptors denoted GABAA amino acids, glycine, and substance P (ref. 17 and unpub- and GABAB. Using Xenopus oocytes, we compared the elec- lished results). Herein we report on the GABA receptors trical and pharmacological properties of GABA receptors encoded by retina RNAs. expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA re- sponses with features characteristic of currents mediated by MATERIALS AND METHODS GABAA receptors. In contrast, RNA from retina expressed Eleven poly(A)+ RNA preparations were made from bovine responses mediated by GABAA receptors and, in addition, retina, 2 preparations were from rat cerebral cortex, and 1 GABA responses that were insensitive to the GABAA antago- preparation was from bovine cerebral cortex. Nine bovine nist bicuculline and the GABAB agonist baclofen and showed no and 1 modulation by barbiturates or benzodiazepines. The bicucul- retina RNA preparations, 1 bovine cortex preparation, line/baclofen-insensitive GABA response was a Cl current rat cortex preparation were made using the phenol/ that was blocked by picrotoxin but showed little desensitization chloroform procedure (18). Two bovine retina RNA prepa- or outward rectification. Our results suggest that mmalian rations and 1 rat cortex preparation were made using the acid retina contains RNAs encoding GABA receptors with distinct guanidinium thiocyanate method (19). Xenopus oocytes, at pharmacology. stages V and VI of development, were microinjected with approximately 100 ng of total poly(A)+ RNA in 50 n1. Five bovine retina RNA preparations were size-fractionated on In mammals, there are two well-characterized classes of (20). Oocytes receptors for the inhibitory neurotransmitter y-aminobutyric 10-30%o (wt/vol) sucrose density gradients acid (GABA). GABAA receptors are ligand-gated Cl- chan- were injected with approximately 25 ng of fractionated nels that are competitively antagonized by bicuculline, non- poly(A)+ RNA, in 50 nl. Two days after injection, enveloping competitively blocked by picrotoxin, and allosterically mod- ovarian tissues were removed by treatment with collagenase ulated by barbiturates and benzodiazepines (1-3). Molecular (21). cloning of cDNAs encoding GABAA receptor subunits indi- Electrical recordings were made using a two-electrode cates that the receptors are heteromeric and comprised of up voltage clamp, in a chamber (0.5 ml) continuously perfused to four different subunits, found in a variety ofclosely related (7-10 ml/min) with frog Ringer solution (115 mM NaCl/2 mM subtypes (e.g., refs. 4-7). In contrast, GABAB receptors KCl/1.8 mM CaCl2/5 mM Hepes, pH 7.0). All drugs were regulate K+ and Ca2+ channels through GTP-binding proteins dissolved in the perfusing Ringer solution. Chloride substi- and intracellular messenger pathways (8). These receptors tutions were made by mixing normal Ringer with the appro- have not been cloned but are presumed to belong to the priate volumes of a low Cl- solution (115 mM sodium superfamily of GTP-binding-protein-coupled receptors. isethionate/2 mM KCl/1.8 mM CaCl2/5 mM Hepes, pH 7.0). GABAB receptors are selectively activated by baclofen, are Zero-Na+ media were 115 mM Tris.HCI/2 mM KCl/1.8 mM antagonized by phaclofen and 2-hydroxysaclofen, and are not CaCl2, pH 7.0, or 115 mM choline chloride/2 mM KCl/1.8 affected by bicuculline, picrotoxin, or any of the GABAA mM CaCl2/5 mM Tris'HCI, pH 7.0. Zero-Ca2+ Ringer was modulators (9, 10). supplemented with 10 mM MgCl2 and 1 mM EGTA. Mem- Xenopus oocytes are now widely used to study receptors brane current responses were recorded from >200 oocytes, and ion channels expressed after microinjection of either taken from >40 frogs, and in all cases uninjected oocytes heterologous poly(A)+ RNA or RNAs transcribed from gave no significant response to GABA. Intraoocyte injections cloned cDNAs (for reviews, see refs. 11 and 12). GABAA of EGTA were made by pneumatic pressure ejection from subunits are readily expressed in oocytes and assemble to micropipettes as described (22). Between 50 and 100 pmol of form receptors that have electrical and pharmacological EGTA were injected, and chelation of intracellular Ca2+ was properties similar to those reported for cells in situ (e.g., refs. confirmed by monitoring abolition of Ca2+-gated Cl- cur- 4-7, 13-15). rents. (+)-Baclofen and 2-hydroxysaclofen were obtained Almost every neurotransmitter/neuromodulator identified from Research Biochemicals (Natick, MA). All other drugs in mammalian brain has also been found in retina (e.g., ref. and reagents were from Sigma. 16). We used Xenopus oocytes to characterize neurotrans- mitter receptors expressed by retina RNAs, investigating whether there were any clear differences between the prop- Abbreviations: GABA, 'y-aminobutyric acid; IG-A. membrane cur- erties of brain and retina receptors. Initial studies, using rent elicited through activation of GABAA receptors; IG-,'t, mem- brane current response elicited by GABA in oocytes injected with retina RNA; IG-BR, bicuculline-resistant GABA response. The publication costs of this article were defrayed in part by page charge *Present address: Dipartimento di Farmacologia, Universita' degli payment. This article must therefore be hereby marked "advertisement" Studi di Firenze, viale G. B. Morgagni 65, 50137 Firenze, Italy. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4318 Downloaded by guest on September 29, 2021 Neurobiology: Polenzani et al. Proc. Natl. Acad. Sci. USA 88 (1991) 4319 RESULTS induced by bovine or rat cortex poly(A)+ RNA (Fig. 2). Maximum responses, elicited by 1-10 mM GABA, were Size-Fractionation of Bovine Retina RNA. At a holding 100-300 nA for IG-ret and 300-3000 nA for IG-A Nevertheless, potential of -60 mV, oocytes injected with total poly(A)+ typical threshold concentrations for eliciting IG-ret were be- RNA from bovine retina responded to GABA with small tween 0.2 and 0.4 IxM GABA, consistently lower than (10-30 nA) inward membrane currents, which we will call thresholds for IG-A (1.0-2.0 /LM). IG-ret showed a steep IG-ret* To increase the size of IG'ret, total poly(A)+ RNA was concentration dependence, with 10 /xM GABA already elic- size-fractionated by sucrose density gradient centrifugation. iting between 75 and 95% of the maximum response. The were oocytes electrically Fractions injected into and assayed re- to determine enrichment for mRNAs encoding GABA recep- concentration of GABA required to elicit half-maximal ± for (n = 21), tors. The fractionation profile for responses elicited by sponses (EC50) was only 1.48 0.32 gM IG-ret as compared to 69.7 ± 14.3 ,uM for induced by rat cortex GABA appeared to be a single peak spread over 3 or 4 IG-A fractions of the 30 fractions collected, corresponding to (n = 21) and 84.6 ± 15.3 .aM for IG-A induced by bovine cortex RNAs of3000-5000 nucleotides. Fractions showing the high- (n = 6). When the dose-response curve for 'G-ret was plotted on the limiting slope value of est activity expressed responses approximately 10-fold larger double-logarithmic coordinates, than total poly(A)+ RNA (i.e., 50-300 nA). induced by the initial rising phase (calculated from the first five data IG-ret points) was 2.69 ± 0.29, indicating a high level of cooper- both fractionated and unfractionated RNAs was then phar- ± macologically and electrically characterized. Using oocytes ativity. The corresponding slope values for 'G6A were 1.60 and ± 0.09 (rat cortex), similar to same was compared with the well- 0.04 (bovine cortex) 1.63 from the frogs, IG-ret (13-15, 25). responses total poly(A)+ those reported in oocytes characterized GABA expressed by GABA Responses. The RNA isolated from bovine or rat cerebral cortex, previously Bicuculline/Baclofen-Insensitive shown to be mediated by GABAA receptors ('G-A) (e.g., refs. clear differences between IG-ret and IG-A in desensitization 13-15, 23-25). Most of the following experiments show data and dose-response relationship prompted a more detailed obtained from oocytes injected with fractionated bovine characterization of the GABA response expressed by retina RNA. by bovine and rat cortex RNA retina RNA. However, it was clear that size fractionation did poly(A)+ IG-A induced was potently blocked by bicuculline, a specific GABAA not obviously alter any of the basic properties of (e.g., IG-ret (see also refs. 13-15, 25). Inhibition was compet- desensitization, dose-response characteristics, or sensitivity antagonist to antagonists and modulators) but simply increased the itive, with 100 ,uM bicuculline raising response thresholds from 2 to about 200 GABA (Fig. 3A). In striking response amplitude. gM ,tM Desensitization of GABA Responses. In agreement with the contrast, IG-ret was only partially blocked by 1-100 ,tM studies mentioned above, IG-A showed marked desensitiza- bicuculline (Fig. 3B). A major component of the response, >10,M. For example, in the constituting 65-95% of the peak current, was essentially tion with GABA concentrations unaffected the antagonist and even threshold responses, case of by bovine cortex poly(A)+ RNA, by IG-A expressed elicited 0.2-0.4 were not blocked responses elicited by 1 mM GABA desensitized by 50% by tkM GABA, appreciably by 1-100 ,M bicuculline.
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