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Functional and pharmacological properties of GABAρ1Δ51 receptors
Neuroscience Research 36 (2000) 141 – 146 www.elsevier.com/locate/neures
Functional and pharmacological properties of GABAr1D51 receptors Angelo Demuro, Atau´lfo Martı´nez-Torres, Ricardo Miledi * Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Beha6ior, Uni6ersity of California, Ir6ine, CA 92697, USA Received 1 October 1999; accepted 9 November 1999
Abstract g-Aminobutyrate is the main inhibitory neurotransmitter in the vertebrate brain, and the g-aminobutyric acid (GABA) receptor subunit GABAr1D51 is an alternatively spliced form of the GABAr1 receptor that was recently isolated from human retina cDNA libraries. The r1D51 receptor subunit lacks 17 amino acids in the extracellular N-terminal domain and, when expressed in Xenopus oocytes, forms functional homomeric GABA receptors. Unexpectedly, even after a such a big deletion, the fundamental properties of the deleted variant receptors are very similar to those of the complete GABAr1 receptors. For example, both types of receptors are bicuculline resistant, desensitize very little, and are negatively modulated by Zn2 + and positively modulated by La3 + . In spite of such similarities, the GABAr1D51 receptors are more sensitive to GABA, to the specific GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and to Zn2 + , than the complete GABAr1 receptors. The GABAr1D51 receptors extend the variety of inhibitory receptors in the retina. Their functional significance still remains to be determined. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alternative splicing; GABA receptors; GABAC; Inhibitory receptors; Xenopus oocyte
1. Introduction In the adult vertebrate central nervous system, inhibitory synaptic actions are effected mainly by the neurotransmitter g-aminobutyric acid (GABA). This amino acid exerts its effects by acting on membrane receptors that belong to at least two gene families: GABAA and GABAB (Bowery, 1999; Mehta and Ticku, 1999). More recently, a third class of GABA receptors, namely GABAC, were electrophysiologically and pharmacologically defined by expressing bovine retina mRNA in Xenopus oocytes (Polenzani et al., 1991; Woodward et al., 1992a,b,c, 1993, 1994). Molecular cloning showed that GABAC receptors are members of the ionotropic family of receptors and, thus far, at least three different genes (r1 – r3) have been isolated from human (Cutting et al., 1991; Calvo, et al., 1994; Wang et al., 1994), rat (Ogurusu and Shingai, 1996), mouse (Greka et al., 1998) and perch (Qian et al., 1999) * Corresponding author. Tel.: +1-949-824-4730; fax: + 1-949-8243522. E-mail address: [email protected] (R. Miledi)
retinas. The neurotransmitter receptors coded by these genes share many physiological and pharmacological characteristics. For example, and in sharp contrast with GABAA receptors which are made up of more than one type of subunits, the r1 subunits are able to form functional ‘homomeric’ receptors that desensitize little after activation by GABA (Shimada et al., 1992; Calvo et al., 1994; Enz and Cutting, 1998; Hackam et al., 1998). In contrast again to GABAA receptors, GABAC receptors are not blocked by bicuculline and are not potentiated by pentobarbital (Polenzani et al., 1991; Woodward et al., 1992a,b,c, 1993, 1994). Furthermore, co-injection of the r1 and r2 subunits into Xenopus oocytes results in the expression of functional GABA receptors with distinct pharmacology. This suggests that different r subunits are able to form ‘heteromeric’ GABA receptors and thus increase the variety of functional GABAr receptors (Enz and Cutting, 1999). Moreover, (1,2,5,6-tetrahydropyridin-4yl)methylphosphinic acid (TPMPA), the first selective antagonist for GABAC receptors, was designed and found to be 100 times more potent in antagonizing human ‘homomeric’ r1 receptors than rat brain
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GABAA receptors (Murata et al., 1996; Ragozzino et al., 1996), and eight times less potent on r2 receptors (Chebib et al., 1998). Recently, an alternatively spliced form of GABAr1 was cloned from human cDNA libraries and expressed in Xenopus oocytes (Martinez-Torres et al., 1997, 1998a,b). This shorter form lacks 51 nucleotides (eliminating from codon 35 to 51) at the putative extracellular amino-terminal domain of the receptor. This subunit, namely GABAr1D51, is able to form functional homomeric receptors whose properties resemble those of the full GABAr1 receptors. The present study compares the functional characteristics of homomeric human r1 and r1D51 receptors. A preliminary report on some of this work has already appeared (MartinezTorres et al., 1998a).
2. Materials and methods
2.1. Preparation of cDNAs and electrophysiology The coding regions of GABAr1 and GABAr1D51 (Martinez-Torres et al., 1997, 1998b) were shuttled into the expression plasmid pcDNA3 using the restriction sites BamH1 and Xho1 flanking both cDNAs. Recombinant plasmids were purified by the alkaline lysis method and used for nuclear injection into Xenopus oocytes. Xenopus lae6is frogs were anaesthetized (by immersion in ice-cold water) and stages IV – VI oocytes were manually isolated from pieces of ovary by removing the external layers, epithelium and theca (Miledi et al., 1982). The oocytes were enzymatically defolliculated (collagenase type IA) (Miledi and Woodward, 1989) and then kept in Barth’s medium (NaCl, 88 mM; KCl, 1 mM; Ca(NO3)2, 0.33 mM; CaCl2, 0.41 mM; MgSO4, 0.82 mM; NaHCO3, 2.4 mM; Hepes, 5 mM) containing 0.1 mg/ml gentamicin sulfate. The next day, 5 –10 nl plasmid (0.5 mg/ml) were injected into the nucleus. Recordings were obtained 2 – 5 days later from oocytes placed in a 100 ml chamber, impaled with two glass microelectrodes (0.5 – 5 MV) filled with 3 M KCl and clamped at −60 mV. All recordings were made at room temperature (20 – 23°C) in a chamber continuously perfused with Ringer’s solution (NaCl, 115 mM; KCl, 2 mM; CaCl2, 1.8 mM; Hepes, 5 mM; pH 7.4) at a flux of 8–12 ml/min. All drugs were purchased from Sigma, except for TPMPA, which was synthesized as previously described (Murata et al., 1996). Solutions were kept frozen as concentrated stocks in water (0.1–1 M), except for b-alanine and glycine, which were prepared each day by dissolving directly in Ringer solution at the required concentrations. The pH of each solution was always checked and, if necessary, adjusted to pH 7.0.
2.2. Statistical analysis Membrane currents are expressed as percentages of the control responses obtained with GABA alone. A control GABA response was obtained before and after each drug application to account for possible shifts in the amplitude of control currents. The GABA currents, expressed as a function of agonist concentration x were fitted with the four-parameter logistic equation I(x)= Imax/1+Kd/x nH, where Imax is the maximum current, Kd is the dose that activates 50% of the maximum current and nH is the Hill coefficient. Kd values are expressed as mean9S.E.M. of the mean.
3. Results As described previously (Martinez-Torres et al., 1997, 1998a,b), oocytes expressing either human cloned GABA r1 or r1D51 receptors produced very similar GABA currents, and their amplitudes were well maintained, even at high concentrations of GABA. Maximal currents elicited by 10–30 mM GABA were 2–10 mA for both r1 and r1D51 receptors, and the threshold concentrations for eliciting GABA currents in both types of oocytes were typically between 30 and 50 nM GABA. As shown in Fig. 1A and Table 1, the concentrations of GABA required to elicit half-maximal responses (Kd), were different for the two types of receptors: 1.129 0.1 mM (n=7) for r1 receptors, and 0.579 0.1 mM (n= 8) for r1D51 receptors. The corresponding Hill values were also different: 1.690.3 and 2.19 0.4 for r1 and r1D51, respectively. The novel and selective GABAC antagonist TPMPA alone, up to 100 mM, did not consistently activate membrane currents in oocytes expressing either r1 or r1D51 receptors. Similar to its action on r1 receptors, TPMPA also blocked the r1D51 receptors. In the presence of 0.5 mM GABA, the average IC50 was 1.69 0.2 mM (n= 5) for r1 and 0.790.1 mM (n= 8) for r1D51. In contrast, the corresponding Hill coefficients were practically the same: 1.29 0.1 and 1.29 0.2, for r1 and r1D51 receptors, respectively (Fig. 1B and Table 1).
3.1. Modulation of GABA currents by glycine and b-alanine It has been shown previously that GABAr1 receptors are activated by glycine and the putative neurotransmitter b-alanine, and are modulated by polyvalent ions like zinc and lanthanum (Calvo et al., 1994; Calvo and Miledi, 1995). Therefore, it was of interest to examine whether the missing 17 amino acids of r1D51 affected some of these properties. The receptors expressed by r1D51, like those expressed by r1, responded to glycine and b-alanine by generating non-desensitizing Cl− cur-
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Fig. 1. Effects of GABA and TPMPA on GABAr receptors. (A) Dose – response curves of GABA-induced currents in oocytes expressing GABAr1 or GABAr1D51 receptors. (B) TPMPA inhibition of the currents elicited by 0.5 mM GABA. The currents were normalized to the amplitude of that obtained with GABA alone. For this and subsequent figures, the oocytes were voltage-clamped at −60 mV, data points are the mean9 S.E.M. of the mean, and the n numbers indicate the number of oocytes examined.
rents similar to those elicited by GABA. The amplitude of the maximal responses elicited by GABA, glycine and b-alanine were in the order GABA\ b-alanine \ glycine, and these differences were proportionally maintained for both types of receptors. Typical responses to GABA (3 mM), b-alanine (3 mM) and glycine (30 mM) in oocytes expressing GABA r1 or GABA r1D51 are illustrated in Fig. 2. The glycine dose – response curves gave similar Kd values of 14.1 92 and 12.9 99 mM, and Hill numbers of 1.490.2 and 1.490.1 for r1 and r1D51 receptors, respectively; while for b-alanine, the corresponding Kd values were 180 9 28 and 165 9 24 mM, and the Hill values were 1.2 9 0.2 for both receptors (Fig. 3). Moreover, and as was the case for GABAr1 receptors, the effects of GABA, glycine and b-alanine on oocytes expressing GABAr1D51 receptors were voltage independent (Fig. 4); and the equilibrium potentials for the agonist currents (EGABA = −23 9 1.0 mV (n= 5); Eb-alanine = −22 9 1.4 mV (n= 4); Eglycine = − 2191.0 mV (n= 4)) were all close to the equilibrium potential for Cl− ( − 30 mV) in Xenopus oocytes (Kusano et al., 1982).
3.2. Modulation of GABA currents by zinc and lanthanum As is the case for GABAr1 receptors (Calvo et al., 1994; Martinez-Torres et al., 1998a), the GABAr1D51 currents were inhibited by zinc in a dose-dependent manner, and in a voltage-independent way (Fig. 5). The current elicited by 0.5 mM GABA was completely blocked by 100 mM zinc, and the current amplitude recovered fully after 2 – 4 min washing. Under the conditions of our experiments, the GABA current (GABA, 0.5 mM) inhibition caused by zinc (30 mM) decayed with time constants of 9.5 and 8.3 s for oocytes expressing GABAr1 or -r1D51 receptors, respectively. In contrast, the time constants of GABA-current decay on washing of GABA were 28.9 and 26.8 s, respectively.
The dose-inhibition curves for zinc, in the presence of 0.5 mM GABA, gave IC50 values of 17.59 0.8 and 9.29 0.2 mM for r1 and r1D51 receptors, respectively (Figs. 5 and 6A). The corresponding Hill coefficients were 1.29 0.2 and 19 0.1. It is known that various lanthanides potentiate the currents elicited by activation of GABAr1 receptors (Calvo et al., 1994). Co-application of lanthanum produced a rapid and reversible increase of GABA currents in a concentration-dependent way (Fig. 6B). With Table 1 Effects of different drugs on human r1 receptor isoforms Compound
GABA b-Alanine Glycine Lanthanum
TPMPA Zinc
r1
r1D51
Kd (mM)
Hill no.
Kd (mM)
Hill no.
1.12 90.1 180 928 14100 9 200 190 9 6
1.6 1.2 1.4 1.1
0.5790.1 165 924 12900 9 900 207 97
2 1.2 1.4 0.9
IC50
Hill no.
IC50
Hill no.
1.6 90.2 17.5 90.8
1.2 1.2
0.7 90.1 9.2 90.2
1.2 1
Fig. 2. Currents elicited by GABA, glycine and b-alanine. Sample records comparing the currents evoked by GABA (3 mM), glycine (30 mM) and b-alanine (3 mM) in two different oocytes expressing GABAr1 or GABAr1D51 receptors. Bars indicate the timing of drug applications.
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Fig. 3. Dose – response curves for b-alanine and glycine acting on oocytes expressing r1 or r1D51 receptors. Each point represents the mean 9 S.E.M. of four to six oocytes.
Fig. 4. Current – voltage relationships for GABA, glycine and b-alanine from an oocyte expressing r1D51 receptors. In each curve, the points represent the peak current elicited by the respective agonist and normalized to the current obtained at − 140 mV. The inset shows a sample record with 20 mV steps, from − 140 to + 40 mV, before and during the current induced by glycine (30 mM).
(Enz and Cutting, 1998; Whiting et al., 1999) and are differentially expressed along the central nervous system (Salceda et al., 1993; Enz and Cutting, 1999). Moreover, the coding potential of the GABA receptor genes is further increased by the mechanisms of alternative splicing. For example, mRNAs coding for a6, b4 and g2 subunits alternate exons to give rise to receptors with different functional properties (Bateson et al., 1991; Harvey et al., 1994; Korpi et al., 1994). So far, only two spliced forms of the r1 subunit, named r1D51 and r1D450, have been found in the human retina (Martinez-Torres et al., 1998b); and injection of the r1D51 spliced form into Xenopus oocytes leads to the expression of functional GABA receptors, whereas the r1D450 subunit, so far, has not produced functional receptors. Our results showed that homomeric r1D51 receptors were more sensitive to GABA, and had a higher level of cooperativity, than the full r1 receptors. Moreover, the specific GABAC antagonist TPMPA was also more potent on r1D51 receptors than on r1 receptors. Furthermore, and in agreement with previous data obtained with the full GABAr1 receptor (Calvo and Miledi, 1995), glycine and b-alanine were also direct activators of r1D51 receptors; and there were no significant differences between the Kd values of both types of receptors for b-alanine and for glycine. The agonist order of potency for both types of receptors was GABA\ b-alanine\glycine, while the corresponding Hill coefficients were GABA\ glycine\ b-alanine. All this suggests that while the three agonists open Cl− permeable channels in both types of receptors (Fig. 4; see also Calvo and Miledi, 1995), their gating mechanisms and sites of action may be somewhat different.
3 mM La3 + , the amplitude of the currents elicited by GABA (0.5 mM) acting on the r1D51 receptors increased to 21599% (n = 6) of the control, while the r1 currents increased to 180 95% (n = 5). Moreover, the lanthanum concentration – potentiation curves for 0.5 mM GABA gave Kd of 190 96 and 207 97 mM for GABAr1 and GABAr1D51 receptors, respectively (Fig. 6). The corresponding Hill coefficients were 1.19 0.1 and 0.99 0.1. The potentiation of GABA currents by lanthanum was voltage-independent in both cases (data not shown).
4. Discussion To date, the family of ionotropic GABA receptors consists of at least five different types of subunits (a, b, g, d and r), which have several homologous subunits (a1-6, b1-3, g1-3 and r1-3) encoded by different genes
Fig. 5. Effects of Zn2 + and La3 + as a function of membrane potential on an oocyte expresing GABAr1D51 receptors. The current – voltage relationships are shown in the absence and in the presence of either Zn2 + or La3 + . The oocyte was maintained at a potential of − 60 mV, and brief volage steps were applied from −160 to 60 mV. (Inset) Sample currents elicited by GABA 0.5 mM alone and together with Zn2 + or La3 + .
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Fig. 6. Modulation of GABA currents by Zn2 + and La3 + . (A) Inhibition of GABA (0.5 mM) current by Zn2 + . (B) Potentiation of GABA (0.5 mM) current by La3 + ; in oocytes expressing r1 or r1D51 receptors.
Polyvalent cations are well known to alter the function of ion channels and neurotransmitter receptors (Heuser and Miledi, 1971; Westbrook and Mayer, 1987; Smart, 1992; Calvo et al., 1994; Martinez-Torres et al., 1998a) In particular, zinc has been found to block the currents elicited by activation of GABAr1 receptors (Calvo et al., 1994), and a single residue (His 156) of the r1 receptor has been considered responsible for the inhibitory effect of zinc (Wang et al., 1995). It is interesting that although this particular residue is present in both isoforms of the r1 receptor, there were some differences in the blocking properties of the ion. Moreover, two His residues (His 44 and His 48) are lost within the deleted region of the GABAr1D51. Since these histidine residues are potentially able to interact with zinc ions, it might be predicted that deleting them would change the r1D51 receptor susceptibility to zinc inhibition. In contrast, we found that the IC50 for GABAr1D51 is actually slightly lower than that of GABAr1. It has been known for years that lanthanum ions alter the effects of acetylcholine quanta at the frog neuromuscular junction (Heuser and Miledi, 1971). Furthermore, La3 + acts on GABAA receptors on a site different from the sites of action of barbiturates, benzodiazepines, picrotoxin, or copper/zinc; and in some preparations lanthanides are able to induce a Cl− current by direct activation of GABA receptors (Im et al., 1992; Im and Pregenzer, 1993; Ma and Narahashi, 1993). We did not see an obvious direct channel gating effect of lanthanum on either GABAr1 or GABAr1D51 receptors, but similar to its potentiating action on the r1 receptor (Calvo et al., 1994), lanthanum enhanced also the GABAr1D51 currents, acting as a positive modulator in both cases. It is somewhat surprising that the loss of 17 amino acids in the N-terminal region of the protein did not produce more remarkable differences between the wild and deleted types of receptors. A smaller deletion, of 30 nucleotides (226–255), in the extracellular N-terminal portion of the GABAA a6 subunit, rendered the receptors non-functional (Korpi et al., 1994). This alternative
spliced form of a6 is present at high levels in the rat cerebellum (20% of the a6 mRNAs), and its physiological role is still unknown. The higher sensitivity of GABAr1D51 receptors to GABA, TPMPA, zinc and lanthanum may be due to changes in binding affinities and/or changes in the gating of the channel; and it is possible that these functional differences will help to discriminate between individual r1 receptors in neurons. It is also interesting that loosing the 17 amino acids did not greatly affect the subunit assembly of the homomeric receptors, since the level of expression of r1 and r1D51 receptors were practically the same. It is very likely that GABAC receptors play an important role in the retina as well as in various brain functions. Therefore, it is necessary that we comprehend better the functional and pharmacological properties of the different subtypes of GABAC receptors expressed in the nervous system. The present work is an effort towards that goal.
Acknowledgements This work was supported by the National Science Foundation (IBN 9604499) and by CONACyT, Me´xico (G25775N). A. M.-T. acknowledges support from CONACyT, Me´xico.
References Bateson, A.N., Lasham, A., Darlison, M.G., 1991. g-Aminobutyric acidA receptor heterogeneity is increased by alternative splicing of a novel b-subunit gene transcript. J. Neurochem. 56, 1437–1440. Bowery, N.G., 1999. Metabotropic GABAB receptors. Neurotransmissions 15, 3 – 11. Calvo, D.J., Miledi, R., 1995. Activation of GABA r1 receptors by glycine and b-alanine. Neuroreport 6, 1118 – 1120. Calvo, D.J., Vazquez, A.E., Miledi, R., 1994. Cationic modulation of r1-type g-aminobutyrate receptors expressed in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 91, 12725 – 12729. Chebib, M., Mewett, K.N., Johnston, G.A., 1998. GABAC receptor antagonists differentiate between human r1 and r2 receptors expressed in Xenopus oocytes. Eur. J. Pharmacol. 357, 227–234.
146
A. Demuro et al. / Neuroscience Research 36 (2000) 141–146
Cutting, G.R., Lu, L., O’Hara, B.F., Kasch, L.M., MontroseRafizadeh, C., Donovan, D.M., Shimada, S., Antonarakis, S.E., Guggino, W.B., Uhl, G.R., Kazazian, H.H.J.R., 1991. Cloning of the g-aminobutyric acid GABA r1 cDNA: a GABA receptor highly expressed in the retina. Proc. Natl. Acad. Sci. USA 88, 2673 – 2677. Enz, R., Cutting, G.R., 1998. Molecular composition of GABAC receptors. Vision Res. 38, 1431–1441. Enz, R., Cutting, G.R., 1999. GABAC receptor r subunits are heterogeneously expressed in the human CNS and form homoand heterooligomers with distinct physical properties. Eur. J. Neurosci. 11, 41 – 50. Greka, A., Koolen, J.A., Lipton, S.A., Zhang, D., 1998. Cloning and characterization of mouse GABAC receptor subunits. Neuroreport 9, 229 – 232. Hackam, A.S., Wang, T.L., Guggino, W.B., Cutting, G.R., 1998. Sequences in the amino termini of GABA r and GABAA subunits specify their selective interaction in vitro. J. Neurochem. 70, 40– 46. Harvey, R.J., Chinchetru, M.A., Darlison, M.G., 1994. Alternative splicing of a 51-nucleotide exon that encodes a putative protein kinase C phosphorylation site generates two forms of the chicken g-aminobutyric acidA receptor b2 subunit. J. Neurochem. 62, 10– 16. Heuser, J., Miledi, R., 1971. Effects of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Soc. (Lond. B) 179, 247 – 260. Im, W.B., Pregenzer, J.F., 1993. Interaction of La3 + with GABAA receptors in rat cerebrocortical membranes as detected with [35S]tbutylbicyclophosphorothionate binding. Eur. J. Pharmacol. 245, 111 – 117. Im, M.S., Hamilton, B.J., Carter, D.B., Im, W.B., 1992. Selective potentiation of GABA-mediated Cl− current by lanthanum ion in subtypes of cloned GABAA receptors. Neurosci. Lett. 144, 165 – 168. Korpi, E.R., Kuner, T., Kristo, P., Kohler, M., Herb, A., Luddens, H., Seeburg, P.H., 1994. Small N-terminal deletion by splicing in cerebellar a6 subunit abolishes GABAA receptor function. J. Neurochem. 63, 1167–1170. Kusano, K., Miledi, R., Stinnakre, J., 1982. Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane. J. Physiol. (Lond.) 328, 143–170. Ma, J.Y., Narahashi, T., 1993. Enhancement of g-aminobutyric acidactivated chloride channel currents by lanthanides in rat dorsal root ganglion neurons. J. Neurosci. 13, 4872–4879. Martinez-Torres, A., Vazquez, A.E., Panicker, M.M., Miledi, R., 1997. Cloning and functional expression of alternative spliced variants of the r1 g-aminobutyrate receptor. Pharmacologist 30, 225. Martinez-Torres, A., Demuro, A., Miledi, R., 1998a. Modulation of GABA r1D51 receptors by polyvalent cations. FASEB J. 12, 440. Martinez-Torres, A., Vazquez, A.E., Panicker, M.M., Miledi, R., 1998b. Cloning and functional expression of alternative spliced variants of the r1 g-aminobutyrate receptor. Proc. Natl. Acad. Sci. USA 95, 4019 – 4022. Mehta, A.K., Ticku, M.K., 1999. An update on GABAA receptors. Brain. Res. 29, 196 – 217. Miledi, R., Woodward, R.M., 1989. Effects of defolliculation on membrane current responses of Xenopus oocytes. J. Physiol. (Lond.) 416, 601 – 621. Miledi, R., Parker, I., Sumikawa, K., 1982. Synthesis of chick brain GABA receptors by frog oocytes. Proc. R. Soc. (Lond. B) 216, 509 – 515.
Murata, Y., Woodward, R.M., Miledi, R., Overman, L.E., 1996. The first selective antagonist for a GABAC receptor. Bioorg. Med. Chem. Lett. 6, 2073 – 2076. Ogurusu, T., Shingai, R., 1996. Cloning of a putative g-aminobutyric acid receptor subunit r3 cDNA. Biochem. Biophys. Acta 1305, 15 – 18. Polenzani, L., Woodward, R.M., Miledi, R., 1991. Expression of mammalian g-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 88, 4318 – 4322. Qian, H.H., Dowling, J.E., Ripps, H., 1999. Molecular and pharmacological properties of GABA-rho subunits from white perch retina. J. Neurobiol. 37, 305 – 320. Ragozzino, D., Woodward, R.M., Murata, Y., Eusebi, F., Overman, L.E., Miledi, R., 1996. Design and in vitro pharmacology of a selective g-aminobutyric acidC receptor antagonist. Mol. Pharmacol. 50, 1024 – 1030. Salceda, R., Vasquez, A.E., Miledi, R., 1993. Espression of GABAC receptor mRNA in the vertebrate retinas. Soc. Neurosci. 19, 91. Shimada, S., Cutting, G., Uhl, G.R., 1992. aminobutyric acid A or C receptor? g-Aminobutyric acid r1 receptor RNA induced bicuculline-, barbiturate- and benzodiazepine insensitive g-aminobutyric acid responses in Xenopus oocytes. Mol. Pharmacol. 41, 683 – 687. Smart, T.G., 1992. A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones. J. Physiol. (Lond.) 447, 587 – 625. Wang, T.L., Guggino, W.B., Cutting, G.R., 1994. A novel gaminobutyric acid receptor subunit (r2) cloned from human retina forms bicuculline-insensitive homooligomeric receptors in Xenopus oocytes. J. Neurosci. 14, 6524 – 6531. Wang, T.L., Hackam, A., Guggino, W.B., Cutting, G.R., 1995. A single histidine residue is essential for zinc inhibition of GABA r1 receptors. J. Neurosci. 15, 7684 – 7691. Westbrook, G.L., Mayer, M.L., 1987. Micromolar concentrations of Zn2 + antagonize NMDA and GABA responses of hippocampal neurons. Nature (Lond.) 328, 640 – 643. Whiting, P.J., Bonnert, T.P., McKernan, R.M., Farrar, S., Le Bourdelles, B., Heavens, R.P., Smith, D.W., Hewson, L., Rigby, M.R., Sirinathsinghji, D.J., 1999. Molecular and functional diversity of the expanding GABAA receptor gene family. Ann. N.Y. Acad. Sci. 868, 645 – 653. Woodward, R.M., Polenzani, L., Miledi, R., 1992a. Effects of steroids on g-aminobutyric acid receptors expressed in Xenopus oocytes by poly(A)+ RNA from mammalian brain and retina. Mol. Pharmacol. 41, 89 – 103. Woodward, R.M., Polenzani, L., Miledi, R., 1992b. Effects of hexachlorocyclohexanes on g-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina. Mol. Pharmacol. 41, 1107 – 1115. Woodward, R.M., Polenzani, L., Miledi, R., 1992c. Characterization of bicuculline/baclofen-insensitive g-aminobutyric acid receptors expressed in Xenopus oocytes. I. Effects of Cl− channel inhibitors. Mol. Pharmacol. 42, 165 – 173. Woodward, R.M., Polenzani, L., Miledi, R., 1993. Characterization of bicuculline/baclofen-insensitive (r-like) g-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of gaminobutyric acidA and g-aminobutyric acidB receptor agonists and antagonists. Mol. Pharmacol. 43, 609 – 625. Woodward, R.M., Polenzani, L., Miledi, R., 1994. Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain GABAA receptors expressed in Xenopus oocytes. J. Pharmacol. Exp. Therap. 268, 806 – 817.
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Functional and pharmacological properties of GABAr1D51 receptors Angelo Demuro, Atau´lfo Martı´nez-Torres, Ricardo Miledi * Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Beha6ior, Uni6ersity of California, Ir6ine, CA 92697, USA Received 1 October 1999; accepted 9 November 1999
Abstract g-Aminobutyrate is the main inhibitory neurotransmitter in the vertebrate brain, and the g-aminobutyric acid (GABA) receptor subunit GABAr1D51 is an alternatively spliced form of the GABAr1 receptor that was recently isolated from human retina cDNA libraries. The r1D51 receptor subunit lacks 17 amino acids in the extracellular N-terminal domain and, when expressed in Xenopus oocytes, forms functional homomeric GABA receptors. Unexpectedly, even after a such a big deletion, the fundamental properties of the deleted variant receptors are very similar to those of the complete GABAr1 receptors. For example, both types of receptors are bicuculline resistant, desensitize very little, and are negatively modulated by Zn2 + and positively modulated by La3 + . In spite of such similarities, the GABAr1D51 receptors are more sensitive to GABA, to the specific GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and to Zn2 + , than the complete GABAr1 receptors. The GABAr1D51 receptors extend the variety of inhibitory receptors in the retina. Their functional significance still remains to be determined. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alternative splicing; GABA receptors; GABAC; Inhibitory receptors; Xenopus oocyte
1. Introduction In the adult vertebrate central nervous system, inhibitory synaptic actions are effected mainly by the neurotransmitter g-aminobutyric acid (GABA). This amino acid exerts its effects by acting on membrane receptors that belong to at least two gene families: GABAA and GABAB (Bowery, 1999; Mehta and Ticku, 1999). More recently, a third class of GABA receptors, namely GABAC, were electrophysiologically and pharmacologically defined by expressing bovine retina mRNA in Xenopus oocytes (Polenzani et al., 1991; Woodward et al., 1992a,b,c, 1993, 1994). Molecular cloning showed that GABAC receptors are members of the ionotropic family of receptors and, thus far, at least three different genes (r1 – r3) have been isolated from human (Cutting et al., 1991; Calvo, et al., 1994; Wang et al., 1994), rat (Ogurusu and Shingai, 1996), mouse (Greka et al., 1998) and perch (Qian et al., 1999) * Corresponding author. Tel.: +1-949-824-4730; fax: + 1-949-8243522. E-mail address: [email protected] (R. Miledi)
retinas. The neurotransmitter receptors coded by these genes share many physiological and pharmacological characteristics. For example, and in sharp contrast with GABAA receptors which are made up of more than one type of subunits, the r1 subunits are able to form functional ‘homomeric’ receptors that desensitize little after activation by GABA (Shimada et al., 1992; Calvo et al., 1994; Enz and Cutting, 1998; Hackam et al., 1998). In contrast again to GABAA receptors, GABAC receptors are not blocked by bicuculline and are not potentiated by pentobarbital (Polenzani et al., 1991; Woodward et al., 1992a,b,c, 1993, 1994). Furthermore, co-injection of the r1 and r2 subunits into Xenopus oocytes results in the expression of functional GABA receptors with distinct pharmacology. This suggests that different r subunits are able to form ‘heteromeric’ GABA receptors and thus increase the variety of functional GABAr receptors (Enz and Cutting, 1999). Moreover, (1,2,5,6-tetrahydropyridin-4yl)methylphosphinic acid (TPMPA), the first selective antagonist for GABAC receptors, was designed and found to be 100 times more potent in antagonizing human ‘homomeric’ r1 receptors than rat brain
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GABAA receptors (Murata et al., 1996; Ragozzino et al., 1996), and eight times less potent on r2 receptors (Chebib et al., 1998). Recently, an alternatively spliced form of GABAr1 was cloned from human cDNA libraries and expressed in Xenopus oocytes (Martinez-Torres et al., 1997, 1998a,b). This shorter form lacks 51 nucleotides (eliminating from codon 35 to 51) at the putative extracellular amino-terminal domain of the receptor. This subunit, namely GABAr1D51, is able to form functional homomeric receptors whose properties resemble those of the full GABAr1 receptors. The present study compares the functional characteristics of homomeric human r1 and r1D51 receptors. A preliminary report on some of this work has already appeared (MartinezTorres et al., 1998a).
2. Materials and methods
2.1. Preparation of cDNAs and electrophysiology The coding regions of GABAr1 and GABAr1D51 (Martinez-Torres et al., 1997, 1998b) were shuttled into the expression plasmid pcDNA3 using the restriction sites BamH1 and Xho1 flanking both cDNAs. Recombinant plasmids were purified by the alkaline lysis method and used for nuclear injection into Xenopus oocytes. Xenopus lae6is frogs were anaesthetized (by immersion in ice-cold water) and stages IV – VI oocytes were manually isolated from pieces of ovary by removing the external layers, epithelium and theca (Miledi et al., 1982). The oocytes were enzymatically defolliculated (collagenase type IA) (Miledi and Woodward, 1989) and then kept in Barth’s medium (NaCl, 88 mM; KCl, 1 mM; Ca(NO3)2, 0.33 mM; CaCl2, 0.41 mM; MgSO4, 0.82 mM; NaHCO3, 2.4 mM; Hepes, 5 mM) containing 0.1 mg/ml gentamicin sulfate. The next day, 5 –10 nl plasmid (0.5 mg/ml) were injected into the nucleus. Recordings were obtained 2 – 5 days later from oocytes placed in a 100 ml chamber, impaled with two glass microelectrodes (0.5 – 5 MV) filled with 3 M KCl and clamped at −60 mV. All recordings were made at room temperature (20 – 23°C) in a chamber continuously perfused with Ringer’s solution (NaCl, 115 mM; KCl, 2 mM; CaCl2, 1.8 mM; Hepes, 5 mM; pH 7.4) at a flux of 8–12 ml/min. All drugs were purchased from Sigma, except for TPMPA, which was synthesized as previously described (Murata et al., 1996). Solutions were kept frozen as concentrated stocks in water (0.1–1 M), except for b-alanine and glycine, which were prepared each day by dissolving directly in Ringer solution at the required concentrations. The pH of each solution was always checked and, if necessary, adjusted to pH 7.0.
2.2. Statistical analysis Membrane currents are expressed as percentages of the control responses obtained with GABA alone. A control GABA response was obtained before and after each drug application to account for possible shifts in the amplitude of control currents. The GABA currents, expressed as a function of agonist concentration x were fitted with the four-parameter logistic equation I(x)= Imax/1+Kd/x nH, where Imax is the maximum current, Kd is the dose that activates 50% of the maximum current and nH is the Hill coefficient. Kd values are expressed as mean9S.E.M. of the mean.
3. Results As described previously (Martinez-Torres et al., 1997, 1998a,b), oocytes expressing either human cloned GABA r1 or r1D51 receptors produced very similar GABA currents, and their amplitudes were well maintained, even at high concentrations of GABA. Maximal currents elicited by 10–30 mM GABA were 2–10 mA for both r1 and r1D51 receptors, and the threshold concentrations for eliciting GABA currents in both types of oocytes were typically between 30 and 50 nM GABA. As shown in Fig. 1A and Table 1, the concentrations of GABA required to elicit half-maximal responses (Kd), were different for the two types of receptors: 1.129 0.1 mM (n=7) for r1 receptors, and 0.579 0.1 mM (n= 8) for r1D51 receptors. The corresponding Hill values were also different: 1.690.3 and 2.19 0.4 for r1 and r1D51, respectively. The novel and selective GABAC antagonist TPMPA alone, up to 100 mM, did not consistently activate membrane currents in oocytes expressing either r1 or r1D51 receptors. Similar to its action on r1 receptors, TPMPA also blocked the r1D51 receptors. In the presence of 0.5 mM GABA, the average IC50 was 1.69 0.2 mM (n= 5) for r1 and 0.790.1 mM (n= 8) for r1D51. In contrast, the corresponding Hill coefficients were practically the same: 1.29 0.1 and 1.29 0.2, for r1 and r1D51 receptors, respectively (Fig. 1B and Table 1).
3.1. Modulation of GABA currents by glycine and b-alanine It has been shown previously that GABAr1 receptors are activated by glycine and the putative neurotransmitter b-alanine, and are modulated by polyvalent ions like zinc and lanthanum (Calvo et al., 1994; Calvo and Miledi, 1995). Therefore, it was of interest to examine whether the missing 17 amino acids of r1D51 affected some of these properties. The receptors expressed by r1D51, like those expressed by r1, responded to glycine and b-alanine by generating non-desensitizing Cl− cur-
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Fig. 1. Effects of GABA and TPMPA on GABAr receptors. (A) Dose – response curves of GABA-induced currents in oocytes expressing GABAr1 or GABAr1D51 receptors. (B) TPMPA inhibition of the currents elicited by 0.5 mM GABA. The currents were normalized to the amplitude of that obtained with GABA alone. For this and subsequent figures, the oocytes were voltage-clamped at −60 mV, data points are the mean9 S.E.M. of the mean, and the n numbers indicate the number of oocytes examined.
rents similar to those elicited by GABA. The amplitude of the maximal responses elicited by GABA, glycine and b-alanine were in the order GABA\ b-alanine \ glycine, and these differences were proportionally maintained for both types of receptors. Typical responses to GABA (3 mM), b-alanine (3 mM) and glycine (30 mM) in oocytes expressing GABA r1 or GABA r1D51 are illustrated in Fig. 2. The glycine dose – response curves gave similar Kd values of 14.1 92 and 12.9 99 mM, and Hill numbers of 1.490.2 and 1.490.1 for r1 and r1D51 receptors, respectively; while for b-alanine, the corresponding Kd values were 180 9 28 and 165 9 24 mM, and the Hill values were 1.2 9 0.2 for both receptors (Fig. 3). Moreover, and as was the case for GABAr1 receptors, the effects of GABA, glycine and b-alanine on oocytes expressing GABAr1D51 receptors were voltage independent (Fig. 4); and the equilibrium potentials for the agonist currents (EGABA = −23 9 1.0 mV (n= 5); Eb-alanine = −22 9 1.4 mV (n= 4); Eglycine = − 2191.0 mV (n= 4)) were all close to the equilibrium potential for Cl− ( − 30 mV) in Xenopus oocytes (Kusano et al., 1982).
3.2. Modulation of GABA currents by zinc and lanthanum As is the case for GABAr1 receptors (Calvo et al., 1994; Martinez-Torres et al., 1998a), the GABAr1D51 currents were inhibited by zinc in a dose-dependent manner, and in a voltage-independent way (Fig. 5). The current elicited by 0.5 mM GABA was completely blocked by 100 mM zinc, and the current amplitude recovered fully after 2 – 4 min washing. Under the conditions of our experiments, the GABA current (GABA, 0.5 mM) inhibition caused by zinc (30 mM) decayed with time constants of 9.5 and 8.3 s for oocytes expressing GABAr1 or -r1D51 receptors, respectively. In contrast, the time constants of GABA-current decay on washing of GABA were 28.9 and 26.8 s, respectively.
The dose-inhibition curves for zinc, in the presence of 0.5 mM GABA, gave IC50 values of 17.59 0.8 and 9.29 0.2 mM for r1 and r1D51 receptors, respectively (Figs. 5 and 6A). The corresponding Hill coefficients were 1.29 0.2 and 19 0.1. It is known that various lanthanides potentiate the currents elicited by activation of GABAr1 receptors (Calvo et al., 1994). Co-application of lanthanum produced a rapid and reversible increase of GABA currents in a concentration-dependent way (Fig. 6B). With Table 1 Effects of different drugs on human r1 receptor isoforms Compound
GABA b-Alanine Glycine Lanthanum
TPMPA Zinc
r1
r1D51
Kd (mM)
Hill no.
Kd (mM)
Hill no.
1.12 90.1 180 928 14100 9 200 190 9 6
1.6 1.2 1.4 1.1
0.5790.1 165 924 12900 9 900 207 97
2 1.2 1.4 0.9
IC50
Hill no.
IC50
Hill no.
1.6 90.2 17.5 90.8
1.2 1.2
0.7 90.1 9.2 90.2
1.2 1
Fig. 2. Currents elicited by GABA, glycine and b-alanine. Sample records comparing the currents evoked by GABA (3 mM), glycine (30 mM) and b-alanine (3 mM) in two different oocytes expressing GABAr1 or GABAr1D51 receptors. Bars indicate the timing of drug applications.
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Fig. 3. Dose – response curves for b-alanine and glycine acting on oocytes expressing r1 or r1D51 receptors. Each point represents the mean 9 S.E.M. of four to six oocytes.
Fig. 4. Current – voltage relationships for GABA, glycine and b-alanine from an oocyte expressing r1D51 receptors. In each curve, the points represent the peak current elicited by the respective agonist and normalized to the current obtained at − 140 mV. The inset shows a sample record with 20 mV steps, from − 140 to + 40 mV, before and during the current induced by glycine (30 mM).
(Enz and Cutting, 1998; Whiting et al., 1999) and are differentially expressed along the central nervous system (Salceda et al., 1993; Enz and Cutting, 1999). Moreover, the coding potential of the GABA receptor genes is further increased by the mechanisms of alternative splicing. For example, mRNAs coding for a6, b4 and g2 subunits alternate exons to give rise to receptors with different functional properties (Bateson et al., 1991; Harvey et al., 1994; Korpi et al., 1994). So far, only two spliced forms of the r1 subunit, named r1D51 and r1D450, have been found in the human retina (Martinez-Torres et al., 1998b); and injection of the r1D51 spliced form into Xenopus oocytes leads to the expression of functional GABA receptors, whereas the r1D450 subunit, so far, has not produced functional receptors. Our results showed that homomeric r1D51 receptors were more sensitive to GABA, and had a higher level of cooperativity, than the full r1 receptors. Moreover, the specific GABAC antagonist TPMPA was also more potent on r1D51 receptors than on r1 receptors. Furthermore, and in agreement with previous data obtained with the full GABAr1 receptor (Calvo and Miledi, 1995), glycine and b-alanine were also direct activators of r1D51 receptors; and there were no significant differences between the Kd values of both types of receptors for b-alanine and for glycine. The agonist order of potency for both types of receptors was GABA\ b-alanine\glycine, while the corresponding Hill coefficients were GABA\ glycine\ b-alanine. All this suggests that while the three agonists open Cl− permeable channels in both types of receptors (Fig. 4; see also Calvo and Miledi, 1995), their gating mechanisms and sites of action may be somewhat different.
3 mM La3 + , the amplitude of the currents elicited by GABA (0.5 mM) acting on the r1D51 receptors increased to 21599% (n = 6) of the control, while the r1 currents increased to 180 95% (n = 5). Moreover, the lanthanum concentration – potentiation curves for 0.5 mM GABA gave Kd of 190 96 and 207 97 mM for GABAr1 and GABAr1D51 receptors, respectively (Fig. 6). The corresponding Hill coefficients were 1.19 0.1 and 0.99 0.1. The potentiation of GABA currents by lanthanum was voltage-independent in both cases (data not shown).
4. Discussion To date, the family of ionotropic GABA receptors consists of at least five different types of subunits (a, b, g, d and r), which have several homologous subunits (a1-6, b1-3, g1-3 and r1-3) encoded by different genes
Fig. 5. Effects of Zn2 + and La3 + as a function of membrane potential on an oocyte expresing GABAr1D51 receptors. The current – voltage relationships are shown in the absence and in the presence of either Zn2 + or La3 + . The oocyte was maintained at a potential of − 60 mV, and brief volage steps were applied from −160 to 60 mV. (Inset) Sample currents elicited by GABA 0.5 mM alone and together with Zn2 + or La3 + .
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Fig. 6. Modulation of GABA currents by Zn2 + and La3 + . (A) Inhibition of GABA (0.5 mM) current by Zn2 + . (B) Potentiation of GABA (0.5 mM) current by La3 + ; in oocytes expressing r1 or r1D51 receptors.
Polyvalent cations are well known to alter the function of ion channels and neurotransmitter receptors (Heuser and Miledi, 1971; Westbrook and Mayer, 1987; Smart, 1992; Calvo et al., 1994; Martinez-Torres et al., 1998a) In particular, zinc has been found to block the currents elicited by activation of GABAr1 receptors (Calvo et al., 1994), and a single residue (His 156) of the r1 receptor has been considered responsible for the inhibitory effect of zinc (Wang et al., 1995). It is interesting that although this particular residue is present in both isoforms of the r1 receptor, there were some differences in the blocking properties of the ion. Moreover, two His residues (His 44 and His 48) are lost within the deleted region of the GABAr1D51. Since these histidine residues are potentially able to interact with zinc ions, it might be predicted that deleting them would change the r1D51 receptor susceptibility to zinc inhibition. In contrast, we found that the IC50 for GABAr1D51 is actually slightly lower than that of GABAr1. It has been known for years that lanthanum ions alter the effects of acetylcholine quanta at the frog neuromuscular junction (Heuser and Miledi, 1971). Furthermore, La3 + acts on GABAA receptors on a site different from the sites of action of barbiturates, benzodiazepines, picrotoxin, or copper/zinc; and in some preparations lanthanides are able to induce a Cl− current by direct activation of GABA receptors (Im et al., 1992; Im and Pregenzer, 1993; Ma and Narahashi, 1993). We did not see an obvious direct channel gating effect of lanthanum on either GABAr1 or GABAr1D51 receptors, but similar to its potentiating action on the r1 receptor (Calvo et al., 1994), lanthanum enhanced also the GABAr1D51 currents, acting as a positive modulator in both cases. It is somewhat surprising that the loss of 17 amino acids in the N-terminal region of the protein did not produce more remarkable differences between the wild and deleted types of receptors. A smaller deletion, of 30 nucleotides (226–255), in the extracellular N-terminal portion of the GABAA a6 subunit, rendered the receptors non-functional (Korpi et al., 1994). This alternative
spliced form of a6 is present at high levels in the rat cerebellum (20% of the a6 mRNAs), and its physiological role is still unknown. The higher sensitivity of GABAr1D51 receptors to GABA, TPMPA, zinc and lanthanum may be due to changes in binding affinities and/or changes in the gating of the channel; and it is possible that these functional differences will help to discriminate between individual r1 receptors in neurons. It is also interesting that loosing the 17 amino acids did not greatly affect the subunit assembly of the homomeric receptors, since the level of expression of r1 and r1D51 receptors were practically the same. It is very likely that GABAC receptors play an important role in the retina as well as in various brain functions. Therefore, it is necessary that we comprehend better the functional and pharmacological properties of the different subtypes of GABAC receptors expressed in the nervous system. The present work is an effort towards that goal.
Acknowledgements This work was supported by the National Science Foundation (IBN 9604499) and by CONACyT, Me´xico (G25775N). A. M.-T. acknowledges support from CONACyT, Me´xico.
References Bateson, A.N., Lasham, A., Darlison, M.G., 1991. g-Aminobutyric acidA receptor heterogeneity is increased by alternative splicing of a novel b-subunit gene transcript. J. Neurochem. 56, 1437–1440. Bowery, N.G., 1999. Metabotropic GABAB receptors. Neurotransmissions 15, 3 – 11. Calvo, D.J., Miledi, R., 1995. Activation of GABA r1 receptors by glycine and b-alanine. Neuroreport 6, 1118 – 1120. Calvo, D.J., Vazquez, A.E., Miledi, R., 1994. Cationic modulation of r1-type g-aminobutyrate receptors expressed in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 91, 12725 – 12729. Chebib, M., Mewett, K.N., Johnston, G.A., 1998. GABAC receptor antagonists differentiate between human r1 and r2 receptors expressed in Xenopus oocytes. Eur. J. Pharmacol. 357, 227–234.
146
A. Demuro et al. / Neuroscience Research 36 (2000) 141–146
Cutting, G.R., Lu, L., O’Hara, B.F., Kasch, L.M., MontroseRafizadeh, C., Donovan, D.M., Shimada, S., Antonarakis, S.E., Guggino, W.B., Uhl, G.R., Kazazian, H.H.J.R., 1991. Cloning of the g-aminobutyric acid GABA r1 cDNA: a GABA receptor highly expressed in the retina. Proc. Natl. Acad. Sci. USA 88, 2673 – 2677. Enz, R., Cutting, G.R., 1998. Molecular composition of GABAC receptors. Vision Res. 38, 1431–1441. Enz, R., Cutting, G.R., 1999. GABAC receptor r subunits are heterogeneously expressed in the human CNS and form homoand heterooligomers with distinct physical properties. Eur. J. Neurosci. 11, 41 – 50. Greka, A., Koolen, J.A., Lipton, S.A., Zhang, D., 1998. Cloning and characterization of mouse GABAC receptor subunits. Neuroreport 9, 229 – 232. Hackam, A.S., Wang, T.L., Guggino, W.B., Cutting, G.R., 1998. Sequences in the amino termini of GABA r and GABAA subunits specify their selective interaction in vitro. J. Neurochem. 70, 40– 46. Harvey, R.J., Chinchetru, M.A., Darlison, M.G., 1994. Alternative splicing of a 51-nucleotide exon that encodes a putative protein kinase C phosphorylation site generates two forms of the chicken g-aminobutyric acidA receptor b2 subunit. J. Neurochem. 62, 10– 16. Heuser, J., Miledi, R., 1971. Effects of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Soc. (Lond. B) 179, 247 – 260. Im, W.B., Pregenzer, J.F., 1993. Interaction of La3 + with GABAA receptors in rat cerebrocortical membranes as detected with [35S]tbutylbicyclophosphorothionate binding. Eur. J. Pharmacol. 245, 111 – 117. Im, M.S., Hamilton, B.J., Carter, D.B., Im, W.B., 1992. Selective potentiation of GABA-mediated Cl− current by lanthanum ion in subtypes of cloned GABAA receptors. Neurosci. Lett. 144, 165 – 168. Korpi, E.R., Kuner, T., Kristo, P., Kohler, M., Herb, A., Luddens, H., Seeburg, P.H., 1994. Small N-terminal deletion by splicing in cerebellar a6 subunit abolishes GABAA receptor function. J. Neurochem. 63, 1167–1170. Kusano, K., Miledi, R., Stinnakre, J., 1982. Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane. J. Physiol. (Lond.) 328, 143–170. Ma, J.Y., Narahashi, T., 1993. Enhancement of g-aminobutyric acidactivated chloride channel currents by lanthanides in rat dorsal root ganglion neurons. J. Neurosci. 13, 4872–4879. Martinez-Torres, A., Vazquez, A.E., Panicker, M.M., Miledi, R., 1997. Cloning and functional expression of alternative spliced variants of the r1 g-aminobutyrate receptor. Pharmacologist 30, 225. Martinez-Torres, A., Demuro, A., Miledi, R., 1998a. Modulation of GABA r1D51 receptors by polyvalent cations. FASEB J. 12, 440. Martinez-Torres, A., Vazquez, A.E., Panicker, M.M., Miledi, R., 1998b. Cloning and functional expression of alternative spliced variants of the r1 g-aminobutyrate receptor. Proc. Natl. Acad. Sci. USA 95, 4019 – 4022. Mehta, A.K., Ticku, M.K., 1999. An update on GABAA receptors. Brain. Res. 29, 196 – 217. Miledi, R., Woodward, R.M., 1989. Effects of defolliculation on membrane current responses of Xenopus oocytes. J. Physiol. (Lond.) 416, 601 – 621. Miledi, R., Parker, I., Sumikawa, K., 1982. Synthesis of chick brain GABA receptors by frog oocytes. Proc. R. Soc. (Lond. B) 216, 509 – 515.
Murata, Y., Woodward, R.M., Miledi, R., Overman, L.E., 1996. The first selective antagonist for a GABAC receptor. Bioorg. Med. Chem. Lett. 6, 2073 – 2076. Ogurusu, T., Shingai, R., 1996. Cloning of a putative g-aminobutyric acid receptor subunit r3 cDNA. Biochem. Biophys. Acta 1305, 15 – 18. Polenzani, L., Woodward, R.M., Miledi, R., 1991. Expression of mammalian g-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 88, 4318 – 4322. Qian, H.H., Dowling, J.E., Ripps, H., 1999. Molecular and pharmacological properties of GABA-rho subunits from white perch retina. J. Neurobiol. 37, 305 – 320. Ragozzino, D., Woodward, R.M., Murata, Y., Eusebi, F., Overman, L.E., Miledi, R., 1996. Design and in vitro pharmacology of a selective g-aminobutyric acidC receptor antagonist. Mol. Pharmacol. 50, 1024 – 1030. Salceda, R., Vasquez, A.E., Miledi, R., 1993. Espression of GABAC receptor mRNA in the vertebrate retinas. Soc. Neurosci. 19, 91. Shimada, S., Cutting, G., Uhl, G.R., 1992. aminobutyric acid A or C receptor? g-Aminobutyric acid r1 receptor RNA induced bicuculline-, barbiturate- and benzodiazepine insensitive g-aminobutyric acid responses in Xenopus oocytes. Mol. Pharmacol. 41, 683 – 687. Smart, T.G., 1992. A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones. J. Physiol. (Lond.) 447, 587 – 625. Wang, T.L., Guggino, W.B., Cutting, G.R., 1994. A novel gaminobutyric acid receptor subunit (r2) cloned from human retina forms bicuculline-insensitive homooligomeric receptors in Xenopus oocytes. J. Neurosci. 14, 6524 – 6531. Wang, T.L., Hackam, A., Guggino, W.B., Cutting, G.R., 1995. A single histidine residue is essential for zinc inhibition of GABA r1 receptors. J. Neurosci. 15, 7684 – 7691. Westbrook, G.L., Mayer, M.L., 1987. Micromolar concentrations of Zn2 + antagonize NMDA and GABA responses of hippocampal neurons. Nature (Lond.) 328, 640 – 643. Whiting, P.J., Bonnert, T.P., McKernan, R.M., Farrar, S., Le Bourdelles, B., Heavens, R.P., Smith, D.W., Hewson, L., Rigby, M.R., Sirinathsinghji, D.J., 1999. Molecular and functional diversity of the expanding GABAA receptor gene family. Ann. N.Y. Acad. Sci. 868, 645 – 653. Woodward, R.M., Polenzani, L., Miledi, R., 1992a. Effects of steroids on g-aminobutyric acid receptors expressed in Xenopus oocytes by poly(A)+ RNA from mammalian brain and retina. Mol. Pharmacol. 41, 89 – 103. Woodward, R.M., Polenzani, L., Miledi, R., 1992b. Effects of hexachlorocyclohexanes on g-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina. Mol. Pharmacol. 41, 1107 – 1115. Woodward, R.M., Polenzani, L., Miledi, R., 1992c. Characterization of bicuculline/baclofen-insensitive g-aminobutyric acid receptors expressed in Xenopus oocytes. I. Effects of Cl− channel inhibitors. Mol. Pharmacol. 42, 165 – 173. Woodward, R.M., Polenzani, L., Miledi, R., 1993. Characterization of bicuculline/baclofen-insensitive (r-like) g-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of gaminobutyric acidA and g-aminobutyric acidB receptor agonists and antagonists. Mol. Pharmacol. 43, 609 – 625. Woodward, R.M., Polenzani, L., Miledi, R., 1994. Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain GABAA receptors expressed in Xenopus oocytes. J. Pharmacol. Exp. Therap. 268, 806 – 817.
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