Proc. Nati. Acad. Sci. USA Vol. 88, pp. 1421-1425, February 1991 Neurobiology y-Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the site (neurotransmitter/mutagenesis/channel/inhibitory) DOLAN B. PRITCHETT*tt AND PETER H. SEEBURGt *Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, PA 19104; and tLaboratory of Molecular Neuroendocrinology, Center for Molecular Biology, University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany Communicated by Erminio Costa, November 13, 1990 ABSTRACT Recombinantly expressed y-aminobutyric benzodiazepine agonists. The a, subunit imparts high-affinity acid type A (GABAA) receptors consisting of a,, P2, and 72 binding for CL 218872 (15, 19), while a2, a3, and a5 subunits subunits contain a binding site for that differs create sites with 10-fold lower affinities for this triazolopy- in its properties from that of ac3%2y2 receptors. Amino acid ridozine (8, 15). Several other compounds, including the substitutions between the GABAA receptor a subunits were benzodiazepines and 2-oxoquazepam (20), the analyzed for their effect on the binding of compounds to the f3-carbolines f8-CCM and 3-CCE (21), and the imidazolpy- benzodiazepine site. By converting ever smaller regions of the ridines , , and AHR-14479 (22, 23) show a3 subunit sequence to that of the a, subunit, we show that a similar pharmacological profiles, with the notable exception single substitution (glycine for glutamic acidj increases the that the three last-named compounds fail to bind to ternary affinity for several compounds approximately 10-fold without receptors containing the a5 subunit (8). changing the affinity for nonselective compounds. Hence, the Since such ternary receptors closely mimic the ligand- identified amino acids may interact directly with the ligand and binding properties and differential localizations of central derme part of the benzodiazepine binding sites in these recep- benzodiazepine sites, one can utilize the well-defined recom- tors. binant receptors to characterize molecular determinants of these clinically important drug targets (6, 15-18). One ap- Benzodiazepines are the most widely prescribed anxiolytics, proach to the study ofreceptor binding and function has been sedatives, muscle relaxants, anticonvulsants, and to create chimeric receptors with primary structures derived (1). The benzodiazepines were in clinical use for 15 years from separate receptor subtypes (24). Particular sequence before their site of action was discovered to be the y-ami- changes that alter the binding or signal transduction of the nobutyric acid type A (GABAA) receptor (2). Compounds of, resultant receptor may represent important structure ele- different chemical structure bind at the same site as benzo- ments that confer binding or signal transduction specificity. diazepines and with similar affinity, although they elicit We chose this approach to determine which amino acids different behavioral effects. These compounds include tria- are involved in the differential binding of CL 218872. By zolopyridazines, imidazopyridines, cyclopyrrolones, pyra- assaying the displacement of [3H]Ro 15-1788 (a nonselective zoloquinolinones, and /3-carbolines (3). GABA is the major benzodiazepine antagonist) binding and by mutating the inhibitory neurotransmitter in the brain, and its receptors are low-affinity form to the high-affinity form, we hoped to present on a majority of neurons, which perhaps explains the eliminate changes of a nonspecific nature. We found that a wide range of effects of benzodiazepines and other com- single amino acid change, from a glutamate to a glycine at pounds acting at that site. The GABAA receptor contains an position 225 in the a3 amino acid sequence, substantially intrinsic chloride channel whose gated activity most often increased the affinity of the a3 subunit-containing receptor hyperpolarizes the cell and inhibits excitation. Benzodiaz- for CL 218872. A slightly larger shift resulted from changing epines, in the presence of GABA, increase GABAA receptor an additional three adjacent residues. The altered binding was chloride channel activity and inhibitory tone throughout the observed for several other selective compounds tested. central nervous system (4). Molecular cloning has led to the characterization of nu- merous sequence-related subunits of the GABAA receptor, MATERIALS AND METHODS including six a subunits (5-9), three f8 subunits (10), two y Construction of Chimeric Receptors. Conserved Nco I subunits (11, 12), and a 8 subunit (13). Recombinant coex- restriction sites occur in homologous positions in the rat a, pression of these subunits has produced receptor channels and a3 subunit cDNAs. By cleavage at these sites, a fragment that differ in their pharmacological (6, 8, 14, 15) and biophys- was removed from the a1 cDNA and then used to replace the ical (16-18) properties, suggesting that the heterogeneity of homologous fragment in the rat a3 cDNA (chimera 3). Partial natural GABAA receptors exceeds previous estimates based digests with Nco I allowed the exchange of larger fragments on ligand binding studies in brain membranes. Although the encoding N- or C-terminal domains (chimeras 1 and 2) into true extent of this heterogeneity is unknown, it is important the appropriate positions. All these constructs were made in to note that ternary receptors reconstituted from a, /3, and y the pBluescript phagemid (Stratagene), which lacks Nco I subunits have binding sites for benzodiazepines and channel sites. The chimeric cDNAs were then subcloned into the activities that can be modulated by these compounds (11, 12). pCDM8 expression vector (25). If such ternary receptors contain a y2 subunit, the a subunit Oligonucleotide-Mediated Mutagenesis. Site-directed muta- variant primarily determines the affinity profile of the ben- genesis was performed as described (26) by using the oligo- zodiazepine site (6, 8, 15). Thus, the a6 subunit defines a nucleotides 5'-GCGTGAGCCATCCTCAGCTACTTC- receptor that binds Ro 15-4513 but not or other CAC-3' (Ml); 5'-TCCTGTACTAGACTGGACTATCCCT-

The publication costs of this article were defrayed in part by page charge Abbreviations: GABA, y-aminobutyric acid; GABAA receptor, type payment. This article must therefore be hereby marked "advertisement" A GABA receptor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. fTo whom reprint requests should be addressed.

1421 Downloaded by guest on September 30, 2021 1422 Neurobiology: Pritchett and Seeburg Proc. Natl. Acad. Sci. USA 88 (1991) GTGTCAACAACATGACCAAG-3' (M2); 5'-AGATTT- constructed by exchanging homologous DNA restriction GTTCTTCTCACGAGTCCAITCATAAATTACTTCAGC- fragments between these cDNAs (Fig. 1). Expression con- TCTGGTATAGGCATA-3' (M3); 5'-TGTACTAGACTG- structs carrying the chimeric sequences were used along with GATTATC-3' (M20); 5'-ACTAGACCGGACTATCTCT-3' expression constructs encoding the rat Y2 and the rat 132 (M21); 5'-GGATTATCCCTGTCCCAAC-3' (M22); 5'- subunits for transient transfection of subconfluent 293 cells. TATCTCTGTGTCAACAACATG-3' (M23), and, as a tem- Membranes from these cells were incubated with [3H]Ro plate, single-stranded phagemid DNA containing the rat a3 15-1788 at a concentration equivalent to its Kd (1 nM), and subunit cDNA sequence. Bacterial colonies transformed the displacement of that binding was examined by including with mutagenized DNA were screened by hybridization with results of the oligonucleotides used for mutagenesis. Plasmid DNA various concentrations ofthe displacing ligand. The isolated from hybridizing colonies was sequenced, and the experiments using CL 218872 to displace [3H]Ro 15-1788 correct DNA was used for construction ofeukaryotic expres- (Fig. 1) indicated that the extracellular portion of the a sion plasmids. subunit was responsible for the distinctive pharmacological DNA Transfection in Cultured Cells. Cloned cDNAs en- profile of the particular receptor subtype, although the si- coding the rat ax, 82, and Y2 subunits were subcloned into the multaneous expression of all a, 13, and 'y subunits was pCDM8 expression vector (Invitrogen, San Diego, CA) by necessary for binding of benzodiazepines. standard procedures. Various amounts of5' (100-500 bp) and The comparison of the amino acid sequences of the a,, a2, 3' (100-2000 bp) untranslated regions were included for and a3 subunits from species expressing high- and low- convenience in the subcloning and did not noticeably affect affinity CL 218872 binding sites (rat, bovine, and human) the results. DNAs were combined in equal amounts and reveals clusters of amino acid substitutions in the extracel- added in transfection buffer (27) to exponentially growing lular domain of the a subunit variants (ref. 5; P.H.S., human embryonic kidney 293 cells (ATCC CRL 1573) (14). unpublished data). Particular positions in which the a, sub- After an overnight incubation, medium was removed, and the unit sequences differ from both the a2 and a3 subunit se- cells were washed once with serum-free medium, and then quences were considered likely candidates for residues af- refed serum-containing medium. fecting binding affinities ofselective benzodiazepine receptor Membrane Preparation and Binding Experiments. Cells compounds. Three such clusters were targeted for mutagen- were harvested from plates 48 hr after transfection by re- esis. Oligonucleotides were constructed to separately mutate peated washing with phosphate-buffered saline followed by these amino acid clusters in the a3 subunit to the correspond- pelleting and freezing at -70°C or by immediate homogeni- ing sequences in the a1 subunit (Fig. 2). Membranes of cells zation in 10 mM potassium phosphate buffer (pH 7.2). After transiently expressing each of the mutated a subunits in two subsequent washes, the cell membrane pellet from combination with ,B2 and )y2 subunits were assayed for the centrifugation at 100,000 x g was homogenized in 10 mM binding of [3H]Ro 15-1788. The displacement of that binding potassium phosphate, pH 7.2/100 mM KCI. Incubation mix- was also assayed and a K, value for CL 218872 was deter- tures contained 800 ,ul ofmembranes (derived from 106 cells), mined for each of the mutated receptors. Only one of the 100 ,ul of tritiated ligand ([3H]Ro 15-1788, 75 Ci/mmol, New three mutated receptors (a3M2, Fig. 2) showed binding that England Nuclear; 1 Ci = 37 GBq), and 100 ,ul of various differed from the native a3 subunit-containing receptor. This concentrations of competing ligands. Nonspecific binding mutant showed a 10-fold increase in affinity for CL 218872 was determined in the presence of 1 ,uM . After relative to that seen for the a3 subunit. The Ki value for that incubation for 60 min at 4°C, the membranes were collected displacement was similar to that observed for receptors by rapid filtration on Whatman GF/C filters. Radioactivity containing the a1 subunit. This indicates that the substitution remaining after two subsequent washes with 5 ml of 10 mM offour amino acids within the a3 subunit raises the affinity for potassium phosphate (pH 7.2) was determined by liquid CL 218872 of ternary receptors containing this mutated scintillation spectroscopy with =40% efficiency. subunit to a level equivalent to that of receptors containing the native a1 subunit. To examine ifall four amino acids contribute to this affinity RESULTS change, oligonucleotides were constructed to singly alter A series of chimeric cDNAs containing portions of coding each ofthe four amino acids in mutant a3M2. DNAs encoding sequences for the rat a1 subunit or the rat a3 subunit were these mutated a3 subunits, a3M20, a3M21, a3M22, and A B r3H]jRo 15-1 788 CL 218872 NH2 COOH t. nM K, nM Oc3 L 0.72±0.22 16204-270 xm 0.86±0.1 1 580:-230 N1 0.64±0.2 123- 26 AU] 0.59±0.1 129- 18

...... 3.-E 0.49±0.24 108t 28

55 amino acids FIG. 1. Chimeric receptors and their affinities for [3H]Ro 15-1788 and CL 218872. (A) Graphic representation of the sequence encoding the rat al and a3 subunits and three chimeric subunits ofthe GABAA receptor. Sequences ofthe a1 subunit are unshaded and those ofthe a3 subunit are hatched. The solid areas represent the sequence encoding putative membrane spanning areas. (B) The affinity (Kd or Kj) of a nonselective benzodiazepine, [3H]Ro 15-1788, and a selective triazolopyridazine, CL 218872, for receptors assembled from a wild-type or chimeric a subunit (shown) along with P and y subunits. The numbers indicate the Kd or the Ki (28) means in nM, with the SEM derived from at least three experiments. Downloaded by guest on September 30, 2021 Neurobiology: Pritchett and Seeburg Proc. Natl. Acad. Sci. USA 88 (1991) 1423 A for the a3M2 mutant subunit-containing receptor, but this 3 1 difference was not statistically significant. Binding to recep- 151 * * *** tors containing the other a3 mutants a3M20, a3M21, and al HACPLKFGSYAYTRAEVVYEWTREPARSVVVAEDGSRL a3M23 was indistinguishable from that seen with the a3 a2 S TS T YNASD 0 P subunit itself. These results indicate that the single amino a3 A T A V S LGKNK E 0 acid change from a glutamic acid to a glycine is primarily 2 responsible for conferring high-affinity binding on a previ- ~~227*~~~~~~~ ously low-affinity receptor. NOYDLLGOTVDSGIVOSSTGEYVVMTTHFH LKRKRTK Several other compounds bind with differential affinity to P )S IOKETI K T A- the a3 and a, subunit-containing receptors. The Ki values for L HVVGTEI IR V T- the displacement of [3H]-Ro 15-1788 by some of these com- Cell pounds was determined for mutant a3M22. This mutant Mhembran.e receptor should have increased affinity for a variety of B imidazopyridines (zolpidem, alpidem and AHR-14479) selec- tive for the a, subunit-containing receptor (19, 20). AHR- [3H]RO 15-1788 CL 218872 14479, zolpidem, and alpidem had an affinity more than nM EI, nM 10-fold higher for a3M22P2y2 than for a3,82y2 receptors. The .d, displacement curves in all these experiments were always a3M1 32 0.8.0.2 1726t 105 consistent with a single affinity state (Fig. 4). a3M2f&2y2 0.6*0.1 126±30 DISCUSSION a3M3&y2 0.5±0.2 1830±148 The purpose of these studies was to establish the molecular basis for the different binding affinity of several compounds FIG. 2. Sequence and pharmacological properties of site-specific for two GABAA receptors. Since that difference is based on mutants. (A) Sequence alignment in single letter amino acid code of compounds that bind competitively with the benzodiaz- the relevant portion of the al, a2, and a3 subunits. Mutated areas 1-3 are overlined, and asterisks indicate positions in which amino acid epines, it is reasonable to assume that these pharmacological residues in the a3 subunit were changed to conform to the a, subunit differences would reflect molecular changes in the benzodi- sequence. The numbers 151-227 indicate the position of amino acids azepine binding site. A molecular mapping of this site should in the mature a, sequence. The shaded portion indicates the position be an important step in understanding the actions of benzo- in which the amino acid sequence is thought to enter the lipid bilayer diazepines on the GABAA receptor. One criticism of muta- for the first time. (B) The affinity (Kd or K1) in nM of each of the tional analysis of proteins is that the mutations may have mutant receptors for [3H]Ro 15-1788 and CL 218872. The mean and actions on the protein that are not directly related to the the standard error of the mean are derived from at least three function being assayed. The studies described here were experiments. designed to minimize the objections that could be raised about mutational studies. First, the mutations must leave a3M23 (Fig. 3), were used in transfections and binding assays intact the binding of [3H]Ro 15-1788, the ligand that is being as described above. Only receptors with mutant a3M22 displaced. Hence, any mutation that nonspecifically affected differed significantly in their properties from the a3 subunit- binding would be eliminated from further investigation. Since containing receptor. In this mutant, the affinity for CL 218872 both a1- and a3-containing GABAA receptors bind [3H]Ro was 8-fold greater than in the a3 subunit (Fig. 3). The K, for 15-1788 with similar affinity (15), chimeras made from them CL 218872 displacement of [3H]Ro 15-1788 was consistently should show the same affinity. A second safeguard was that greater than for either the a, subunit-containing receptor or the mutations were designed to increase the binding affinity A for CL 218872. An increase rather than decrease of affinity made it more likely that the observed changes in pharmaco- logical properties were relevant to the structure of the native MUTANT2 receptor. Both of these design criteria were achieved in our a3 DLLG HVVGTEII RSS experiments. Thus, none of the mutations affected the affin- 23 22 21 20 ity of the receptor for [3H]Ro 15-1788, and the relevant al D G VQ mutations clearly increased the affinity for CL 218872. This strongly suggests that the functionally identified amino acid differences between the a1 and the a3 subunit are indeed the B cause of the differing pharmacology. CL 218872 Mutant nM The interpretation that the amino acids involved in the K., selective pharmacology define part of the binding site of 20 1560±248 benzodiazepines is based on two assumptions. The first is 21 1745±270 that compounds bind competitively because of steric overlap, precluding the simultaneous occupation of their respective 22 230 ±16 binding sites. The second is that there is a direct interaction 23 1699 ±190 between the amino acids identified by mutational analysis and the compounds showing altered affinity. While the first FIG. 3. Point mutations in the GABAA receptor a3 subunit and assumption is widely accepted, it is still possible for two affinities for CL 218872. (A) The single letter amino acid code for the compounds to have overlapping, nonisosteric binding sites. a3 subunit in the region defining mutated area 2 (Fig. 2) is shown Using as displacers both triazolopyridazines and imidazopy- along with the amino acid differences between the a, and a3 subunits ridines-compounds with very different chemical struc- in this region (below). The numbered point mutations changing the tures-suggests that the sites for Cr3 sequence into the aC1 sequence are used to identify the individual these compounds have mutants. (B) The Ki value in nM for CL 218872 inhibition of [3H]Ro overlap with the benzodiazepine site. The second assumption 15-1788 binding is shown for each of the mutants. The mean and the is more problematical because a change in amino acid se- standard error of the mean are derived from at least three experi- quence may alter the protein structure at a distant site-e.g., ments. through the destruction of a salt bridge. Further substitutions Downloaded by guest on September 30, 2021 1424 Neurobiology: Pritchett and Seeburg Proc. Natl. Acad. Sci. USA 88 (1991) C A 100 100-

so- 50s 0

* *1- *--- I*-w * ** -- 8 7 6 5 4 8 7 6 5 4 'a AHR-14479, -log M Alpidem, -log M 0 .010* B D 100

50-

8 7 6 5 4 8 7 6 5 4 CL 218872, -log M Zolpidem, -log M

FIG. 4. The inhibition of [3H]Ro 15-1788 binding by CL 218872, AHR-14479, alpidem, and zolpidem. Binding of [3H]Ro 15-1788 to membranes prepared from cells transiently expressing a3, P2, V2 (solid diamonds), or a3m22, (32, Y2 (open squares) receptors is plotted for various concentrations of AHR-14479 (A), CL 218872 (B), alpidem (C), and zolpidem (D) as the competing ligand. The data points are shown in relation to a theoretical sigmoidal curve with a Hill slope of 1 predicted for a single affinity binding site.

for the glutamate residue in the a3 subunit may help to assess the binding of acetylcholine to the nicotinic acetylcholine the effect of the charge change between the glycine residue receptor (32, 33). in the a, subunit and the glutamate residue in the a3 subunit. Since the interactions between ligands and multisubunit It is interesting that selective compounds from two distinct receptor proteins are complex, and since there are variants chemical families seem to interact with the same amino acid. within most subunit classes, molecular determinants for This implies, given the above assumptions, that both types of binding of other compounds at the benzodiazepine site may compounds may align themselves in the binding pocket be distant from those implicated here. For a more complete formed by residues, including that responsible for selectivity picture of the benzodiazepine binding pocket it will be as identified in our experiments. While this may seem un- important to determine the amino acid(s) photolabeled by likely, recent modeling work based on the crystal structure of benzodiazepines. This pocket is likely to be part of more than CL 218872 has suggested that the =N-N= group of the one subunit. Further chimeric receptors constructed from triazole ring may serve the same function as the C=O group other a subunits may also yield more information about the present in almost all benzodiazepine receptor ligands (29). residues important for binding, and electrophysiological ex- Experiments to establish whether the charge on the glutamate periments may even suggest a mechanism by which binding residue is important for selectivity will be necessary before influences ion flux. Chimeric receptors constructed from the determining which charges in the ligand molecules might be diazepam-insensitive a6 subunit and the diazepam-sensitive interacting with this residue. a, subunit may be especially informative in this regard. Pinpointing the residues responsible for drug selectivity can be viewed in the light of work by others on the structure We acknowledge the expert technical assistance of Sabine of the benzodiazepine binding site on GABAA receptors and Grunewald, Anna Herb, and Robert Williamson. We thank Michael the structure of the related nicotinic acetylcholine receptor. B. Robinson for critically reading the manuscript. This work was Farb and coworkers (30, 31) have shown that the funded by grants from Deutsche Forschungsgemeinschaft (SFB Thus, 317/B9) to P.H.S., Children's Seashore House and The Joseph GABAA receptor can be heavily trypsinized and still bind Stokes Research Institute to D.B.P. benzodiazepines. The trypsin cleavage sites can be predicted from the amino acid sequence ofthe receptor subunits. If one 1. Haefely, W., Kyburz, E., Gerecke, M. & Mohler, H. (1985) assumes that those basic amino acids thought to be near the Adv. Drug Res. 14, 165-322. membrane surface where the protein enters the lipid bilayer 2. Costa, E., Guidotti, A. & Mao, C. C. (1975) Adv. Biochem. for the first time are protected from cleavage by steric Pharmacol. 14, 113-130. mechanisms, the next possible cleavage site in the a, subunit 3. Skolnick, P. & Paul S. M. (1987) in GABA and Benzodiazepine occurs 32 amino acids N-terminal (extracellular). Cleavage at Receptors, ed. Squires, R. (CRC, Boca Raton, FL), pp. 91-102. this location would leave the residues identified by our study 4. Olsen, R. & Venter, C., eds. (1987) Benzodiazepine/GABA Receptors and Chloride Channels Structure and Functional membrane-bound. 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