Cationic modulation of pl-type y-aminobutyrate receptors expressed in Xenopus oocytes

A study was made of the effects of diand trivalent cations on homomeric p1-type y-aminobutyrate (GABAp) receptors expressed in Xenopus oocytes after inijection of mRNA coding for the GABA., subunit. GABA elicited large currents with a Kd 1 ,uM. The properties of these GABAA, receptors were similar to those of native bicucullineresistant GABA receptors expressed by retinal mRNA. GABAp, currents showed very little desensitization, were blocked by picrotoxin but not by bicuculline, and were not modulated by barbiturates, benzodiazepines, or j3carbolines. Zn2+ reversibly decreased GABA responses (IC5o = 22 ,uM). Other divalent cations were also tested and their rank order of potency was: Zn2+ Ni2+ Cu2+ >> Cd2+, whereas Ba2+, Co2+, Sr2+, Mn2+, Mg2+, and Ca2+ showed little or no effect. In contrast, La3+ reversibly potentiated the GABA currents mediated by homomeric GABAp, receptors, with an EC5o = 135 ,uM and a maximal potentiation of about 100% (GABA, 1 mM; La3+, 1 mM). Other lanthanides showed similar effects (Lu3+ > Eu3+ > Tb3+ > Gd3+ > Er3+ > Nd3+ > La3+ > Ce3+). Thus, GABAA receptors contain sites for cationic recognition, and in particular, Zn2+ may play a role during synaptic transmission in the retina. Inhibition in the vertebrate brain is mediated mainly by two well-known types of y-aminobutyric acid (GABA) receptors: GABAA and GABAB (1, 2). GABAA receptors are heteromeric complexes, of four or five subunits, that form ligandgated Clchannels (3), whereas GABAB receptors, whose structure is still unknown, regulate K+ and Ca2+ channels through heterotrimeric guanine nucleotide-binding proteins (G proteins) (2). A third type of GABA receptor was discovered by expressing retina mRNA in Xenopus oocytes (4) and its cDNA was isolated from a human retina library (5). Consequently, this receptor is sometimes called GABAretina, GABAp, or GABAC. The physiological and pharmacological properties of the GABAp receptors are very different from those of GABAA or GABAB receptors. For example, GABAp receptors are not blocked by bicuculline, show little desensitization, and are not modulated by barbiturates, benzodiazepines, or other drugs that modulate GABAA receptors (4-18). It has been known for some time that polyvalent cations modulate transmitter action on some receptors. For example, Sr2+ increases the lifetime of the channels gated by acetylcholine at frog endplates (19, 20), and Ca2+ shortens the lifetimes of the channels gated by acetylcholine receptors in frog muscle and of the glutamate receptors in locust muscle (20, 21). More recently, it has been shown that Zn2+, which is present in nerve terminals (22, 23) and is released following nerve stimulation (24, 25), modulates the action of GABAA receptors (25-29). Because of all this we thought it desirable to study the effects ofZn2+ and other polyvalent cations on the function of homomeric GABAp, receptors. This work, carried out in 1992, has been presented in preliminary form (30). MATERIALS AND METHODS Cloning, in Vitro Transcription, and Expression of Homomeric GABAp, Receptors. A 1-kb fragment of the p, gene (the gene encoding the GABAP, receptor) was amplified from a human retina cDNA library (kindly provided by J. Nathans, Johns Hopkins University School of Medicine) by PCR using primers derived from the published P1 sequence (5). The fragment was cloned into pBluescript II KS (Stratagene), labeled by random priming (Boehringer Mannheim kit), and used to screen the library by hybridization to obtain the complete P1 cDNA (A.E.V., M. Panicker, and R.M., unpublished work). P1 cDNA was subcloned into the EcoRI site of pBluescript KS and transcribed in vitro to obtain sense p mRNA. The RNA was microinjected into Xenopus oocytes, and this led to the expression of functional homomeric GABAP1 receptors. Voltage-Clamp Recording of Membrane Currents. Frogs were from Xenopus I (Ann Arbor, MI) or Nasco (Modesto, CA), and Xenopus oocytes were handled as described (31). Briefly, oocytes were injected with 5-50 ng of mRNA (in 50 nl), defolliculated with collagenase, and stored for 3-9 days in Barth's medium with gentamicin at 0.1 mg/ml. Voltageclamp recordings (32) were made from oocytes in a chamber continuously superfused (5-10 ml/min) with frog Ringer solution (115 mM NaCl/2 mM KCl/1.8 mM CaCl2/5 mM Hepes, pH 7.00). Results are presented as the mean ± SEM. All the experiments were carried out at room temperature, 20-23°C. Solutions were made up fresh each day as concentrated (0.001-1 M) stocks in water. Chloride or sulfate salts of the various cations were dissolved in Ringer solution or Ringer solution containing GABA. The pH of each test solution was always checked and adjusted (7.00). All the salts were purchased from Sigma. RESULTS Pharmacological Prorfle of Cloned Homomeric GABA., Receptors. GABAergic agonists and antagonists were assayed on the homomeric GABAp, receptors expressed in oocytes. The results essentially confirmed those previously reported (5, 17) but included additional tests of related compounds. The GABAP, responses were resistant to bicuculline and insensitive to GABAA allosteric modulators such as pentobarbital, diazepam, chlordiazepoxide, flunitrazepam, RO 15Abbreviations: GABA, 'y-aminobutyric acid; TACA, transaminocrotonic acid; CACA, cis-aminocrotonic acid. *Present address: Department of Cell Biology and Anatomy, University of Miami School of Medicine, P.O. Box 016960, N.W. 10th Avenue, Miami, FL 33101. 12725 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 91 (1994) 1788, or methyl, ethyl, or butyl ,B-carboline up to 100 ,M. Furthermore, the rank order for conformational analogs [trans-aminocrotonic acid (TACA) > GABA > muscimol > cis-aminocrotonic acid (CACA)] was as found previously for receptors expressed by native or cloned mRNAs. Currentvoltage relationships were fairly linear (e.g., Fig. 1D) and the reversal potentials (4, 9, 17) were around -20 mV, as expected for currents carried mainly by Clions (33). It is known that cations can modulate GABA-mediated Clcurrents in different types of native or cloned GABAA receptors (34-36). We examined the modulation of GABAP1 receptors by various polyvalent cations. Inhibition of GABAp, Currents by Zn2+. It is known that Zn2+ suppresses GABA responses in a variety of preparations (34-38) and that recombinant GABAA receptors are blocked by Zn2+ (29, 36) with different efficacy. The present experiments were made to determine whether homomeric GABAp receptors were also modulated by Zn2+ ions. Zn2+ inhibited the GABAP, currents in a dose-dependent manner (Fig. 1). At 100 ,M, Zn2+ blocked almost completely the currents elicited by 1 AM GABA, while 30 ,uM Zn2+ reduced them to 33% (IC50 = 21.9 + 1.0 ,uM). The current amplitude recovered fully after short periods (1-3 min) of wash with Zn2+-free Ringer solution. Thus, the blocking effect was dose-dependent (Fig. 1B), and both the onset and decay of Zn2+ action were rapid. Dose-response curves in the absence or presence of 30 AM Zn2+ (Fig. 1C) show that Zn2+ decreased the affinity (Kd) without affecting the maximum response to GABA (in the control, Kd = 1.08 ± 0.36 ,M, Hill coefficient (h) = 2.02 + 0.57; in the presence of Zn2+, Kd = 1.82 ± 0.25 ,uM, h = 1.77 ± 0.43). Furthermore, the inhibition of GABA-induced currents by Zn2+ was strongly dependent on the concentration of GABA, with the