Molecular biology of GABAA receptors.

Molecular

biology

RICHARD

W. OLSEN*

AND

Departments of *phaacology and *Mental

Retardation

of GABAA J. TOBIN1

ALLAN

School of Medicine, and tBiology, *.IBrain Research Institute,

Research Center, University

ABSTRACT The

major

type

transmitter

of receptor

GABAA

receptor, ion

oligomeric

types

f3,

polypeptides

identity

with

and

of

other,

the

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identity. sequence

each

expressed

60-80%

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Key Words: GABAA subtypes . ligand-gated

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the ligand-gated ion channel family of receptors (1, 2). This family of receptors, typified by the nicotinic acetylcholine receptors (3, 4), forms a hetero-oligomeric complex, with the walls of the ion channel contributed from membrane-spanning regions of each of the intrinsic membrane subunits, and the neurotransmitter binding site present on the same protein complex (5-7). Binding of the neurotransmitter (the regulatory ligand) allosterically controls the opening and closing of the ion channel on a millisecond time scale, with no chemical bond breaking or formation (8, 9). In addition to the GABAA receptor/chloride channel, the strychnine-sensitive glycine receptor/chloride channel (10-12) and probably other receptors, e.g., certain excitatory amino acid receptors (13), also appear to be members of this gene superfamily. Molecular cloning technology has allowed deduction of the amino acid sequences of the various polypeptides that are subunits of several of these receptors from numerous tissues and animal species. The subunits making up a specific type of receptor oligomer in a given tissue have a high degree of sequence identity with each other, suggesting that they have evolved from a common ancestral sequence. Each polypeptide subunit in turn exhibits a tissue-dependent heterogeneity that suggests an even more recent common ancestral sequence of closely related but distinct genes. The individual members of each polypeptide family show considerable sequence identity, somewhat less with the other subunit types found in an oligomer, and somewhat less again with the other types of receptors. The most similar sequence domains within any subunit are believed to be those with conserved structural or functional roles, whereas sequence domains with greater sequence variability would be expected to contribute to structural, functional, and tissue diversity. Considerable understanding has been gained regarding the structure and function of the nicotinic acetylcholine receptor family, using both biochemical and molecular cloning approaches. In 1987, Schofield et al. (2) reported the identification and sequences of cDNAs for the a and 3 subunits of the GABAA receptor. These sequences confirmed the relationship to the nicotinic receptors (2) expected from similarities observed in electrophysiological studies (14). Since then, several

‘GABA,

y-aminobutyric

acid;

SDS,

sodium

dodecyl

sulfate.

1469

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groups, especially Seeburg and colleagues (e.g., ref 15), have reported the cloning of multiple types and subtypes of polypeptides, leading to an explosion of information about the GABAA receptors. In this article, we review the latest information on this topic, obtained primarily from molecular biological approaches. We compare GABAA and nicotinic receptor proteins. Further, we will attempt to make some conclusions and speculations about structural and functional properties of the family of oligomeric subtypes of GABAA receptors found in vivo. BIOCHEMICAL

PROPERTIES

The nicotinic receptors from electric organs of electric rays and eels, as well as from vertebrate skeletal muscle, are intrinsic membrane glycoproteins. All are pentameric oligomers, of about 250 kilodaltons (kDa), composed of four different types of subunits, each of approximately 50 kDa, i.e., the subunit composition is a2/3’y#{244} (8, 9). Vertebrate central nervous system nicotinic receptors appear to consist of only a and /3 polypeptides, and may have a total of four or five subunits (4, 7, 16). The oligomeric subtypes vary with developmental stage, as well as with tissue and brain region, and show different pharmacological properties (8). Detailed aspects of the sequences and structural and functional domains are discussed below. GABAA receptors from mammalian brain have been purified by benzodiazepine affinity chromatography and were found to have two major bands on gel electrophoresis in sodium dodecyl sulfate (SDS), the a subunit (53 kDa) and j3 subunit (56 kDa) (17). The native molecular weight (in detergent) was estimated to be between 220 (17) and 355 kDa (18), and the subunit composition was suggested to be a2f32 (19). Photoaffinity labeling of the benzodiazepine binding site with [3H]fiunitrazepam in crude homogenates identified a polypeptide on SDS gels of 51 kDa (20), and a polypeptide band of this size was identified in purified receptor by photolabeling of the 51- to 53-kDa a subunit (17, 21, 22). The major GABA binding site was identified by photolabeling with [3H]muscimol as the 56- to 58-kDa 3 subunit (23). However, both major bands were photolabeled with [3H]fiunitrazepam and [3H]muscimol at high protein concentrations (24), indicating that both subunits may carry both ligand binding sites. Monoclonal antibodies were prepared against partially purified bovine receptor. Individual monoclonal antibodies reacted in Western blots with one or the other (or neither) of two bands in the purified receptor preparation at 51 or 56 kDa (evidently the a and /3 subunits). The two polypeptide antigens colocalized in many brain regions, and both a and j3 subunit-specific

antibodies diazepine

immunoprecipitated binding activities

both GABA and benzo(25, 26). Estimates of the

oligomeric receptor molecular weight by target size irradiation analysis ranged from 220 (27) to 400 kDa (28). Molecular cloning has identified four types of polypeptide (putative subunits) in the molecular weight 1470

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1990

range of 50- to 60-kDa, and multiple genes (subtypes) for each of these types (2, 29-35). The subunit composition in vivo is not known for any tissue but may be deduced from information about the localization of polypeptide subtype mRNAs (29-40), from subtypespecific antibody staining (41), from subtypes defined by ligand binding in tissue section autoradiography (42), and from physically distinct polypeptides detected on SDS gels that show different pharmacological properties by photoaffinity labeling (43, 44). In summary, available evidence suggests that the GABAA receptor is a hetero-oligomer of 220-400 kDa, composed of two to four different polypeptides of about 55 kDa each, and a total of four to five subunits. It is likely that all of the subunits bind GABA and benzodiazepine ligands, probably with different affinities (24, 43, 44). A myriad of oligomeric structures would appear to exist with differential expression in various brain regions. The subtypes differ not only in location, but also in physiological function and mechanisms of biological regulation; it can be assumed that they will also differ in susceptibility to disease processes (but also in sensitivity to pharmacological intervention). STRUCTURE OF THE SUBUNITS DEDUCED GENE SEQUENCES

GABAA FROM

RECEPTOR THE CLONED

Unsuccessful attempts to clone the GABAA receptor included isolation of water-soluble proteolytic fragments identified by photoaffinity labeling with [3H]fiunitrazepam for partial sequencing, NH2-terminal sequencing of purified receptor, identification of expression library peptides with specific antibodies, and expression of fractionated brain mRNA in frog oocytes. The successful approach involved purification of proteolytic fragments from purified bovine brain receptor protein and partial sequencing, followed by oligonucleotide probe hybridization screening of a cloned cDNA library derived from bovine brain (2). Initially, two distinct cDNAs were isolated that contained an open reading frame coding for amino acid sequences that corresponded to the isolated polypeptide fragment sequences. These were called a and /3, apparently under the impression that the purified receptor contained only the two subunit bands (a and /3) indicated by protein staining (17, 45) and immunoblotting (21, 25, 26). Verification of the identity of these two polypeptides as GABAA receptor subunits was provided by expression in Xenopus oocytes of genetically engineered mRNAs corresponding to full-length cDNAs and the production of GABA-regulated chloride channels in the oocytes. At first it appeared that these two polypeptides were necessary and sufficient for full biological activity of the GABAA receptor, including modulation of chloride channel activity by barbiturates and benzodiazepines, picrotoxin and bicuculline, radioligand binding, and cooperative dose-response curves to GABA with appropriate affinity (2). Later, however, it became clear that these expressed receptor/ion channels exhibited

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OLSEN

AND

TOBIN


Figure protein

1. Generic GABAA receptor subunit sequence and putative

topological structure. The numbering follows that of the rat a, sequence (34). Note the NH2 terminal (labeled N, residue 1) presumed extracellular domain, with probable sites for asparagine glycosylation (polymeric black circles at positions 10 and 110), and the cystine bridge (solid line connecting 138 and 152) conserved between all members of the gene superfamily (2). Four putative membranespanning a-helical cylinders Ml, M2,

o

M3, and M4 are shown. The COOHterminus (labeled C, residue 428) is again extracellular. A large intracellular cytoplasmic loop between M3 and M4 is present. The color code indicates the degree of variability (see also Fig. 2) within the family of rat polypeptides published to date: a,, a2, a4, f3, (32, 133, Y2 and (30-34). Those amino acids identical in all the clones are shown in white, those identical in two or more types are gray, those identical in all a but not in (3, -y, orb are black, and those that vary between types are in color (see text for discussion).

Common to All Sequences

a

#{149} Common to Several

most, but not all, of the native properties expected of this protein (29, 30, 46). The isolation of clones for multiple subtypes of each individual polypeptide type (29, 31), plus apparent additional types of putative subunits (30, 32), suggested that the original expression of bovine a1 plus /3 probably did not achieve the actual subunit composition occurring in vivo. Additional a (29) and /3(31) clones from cow and other species were obtained by screening brain cDNA libraries with degenerate oligonucleotide probes whose sequences derived from those of bovine receptor peptides, or the original bovine a and /3 cDNAs. Additional subunit clones were obtained by screening with oligonucleotide probes based on conserved transmembrane segment sequences (30, 32). So far the ‘y and 6 subunits have not been identified as polypeptides in the purified receptor or in brain homogenates. Nor is it known if every receptor oligomer contains each of the polypeptide types, nor how many copies of each type are included. Incorporation of only one of the subtypes of any given subunit type into any one oligomer would be a likely configuration, but it has not been demonstrated. Indeed, the correct in vivo subunit combination is still under investigation, using a variety of techniques, as discussed below. The contribution of the additional polypeptide types to the overall picture is still uncertain. All of the GABAA receptor polypeptide sequences identified to date share sequence identity with one another, including some highly similar domains considered to represent conserved structural motifs. Some

MOLECULAR

BIOLOGY

OF GABAA RECEPTORS

of these are present as well in the nicotinic acetylcholine receptor polypeptides (16). The nicotinic receptor polypeptides and subtypes, in turn, also share sequence elements, and it seems likely that the genes encoding the polypeptides of both receptors are homologous, that is, they have arisen by gene duplication and divergence (47). The generic subunit model shown in Fig. 1 summarizes these similarities. The sequence numbering is based on the rat a1 (32-34). Those residues that are identical in all of the published clones (a, /3, y, 6) are indicated in white, those that are identical in multiple types and subtypes are in gray; those identical only in a polypeptides are black; and those with variable amino acids are red. All of the clones are typified by four putative membrane-spanning domains M1-M4 containing 22-23 ahelical residues with a predominantly hydropathic character, as evaluated by the Kyte and Doolittle program (48). These occur at residues 225-246, 251-272, 284-306, and 394-415 in the COOH-terminal half of the protein. The long hydrophilic NH2-terminal half of the subunit contains potential asparagine-glycosylation sites (two in a1 at positions 10 and 110) and a conserved cysteine pair (shown as a cystine bridge at positions 138-152) believed to participate in ligand binding. There are additional presumed glycosylation sites on other a subtypes and other subunit types. The proteins are indeed glycoproteins (19, 22), and all of the polypeptide sizes on SDS gel electrophoresis are larger than the deduced sequences, suggesting that they are all

1471


glycosylated. All of the published clones encode signal peptide sequences that are presumed to be clipped during the production of mature polypeptide; no NH2terminal sequences have been obtained, possibly due to biological N-acylation (2, 29, 34). The COOH terminus is located at a brief extracellular hydrophilic segment following M4. Between M3 and M4 are long, variable putative intracellular domains that contribute to subtype specificity and participate in intracellular regulatory mechanisms. For example, a consensus sequence shared by substrates for phosphorylation by the cyclic AMPdependent protein kinase is present in 3 but not in a, ‘y, or 6 sequences (2). /3 Subunits have been demonstrated to be phosphorylated in vitro by both protein kinase A (porcine receptor [49]; rat receptor [50]) and protein kinase C (50), the latter phosphorylating a different subtype of f3 than the protein kinase A. In addition, the rat receptor a subunit was phosphorylated in vitro by a previously unknown endogenous kinase that copurified with the receptor on the benzodiazepine affinity column (51). The functional consequences of such GABAA receptor phosphorylation, and the mechanisms of regulation in vivo, are under investigation. As shown in Fig. 1, about 20% sequence identity (50% counting conservative replacements) is observed between the a, /3, and 6 polypeptide types (coded white). The GABA receptor polypeptides have sequences in common with the nicotinic and glycine receptors. Amazingly, more than 10% of GABA receptor amino acid residues are identically positioned in some highly conserved domains in the 20 or so known nicotinic acetylcholine receptor cDNAs (2, 4) and the one glycine receptor cDNA that have been sequenced (10). Residues conserved among all members of the superfamily are located in the membrane-spanning domains and in several segments of the NH2-terminal extracellular regions. This conservation occurs despite the fact that these receptors bind different neurotransmitter ligands and form channels for ions of different charges (cations for acetylcholine, chloride for GABA and glycine). The residues conserved among several GABAA cDNAs (gray [plus white]) include the four transmembrane regions mentioned, which are about 40% identical (80-90% is conservative replacements are also included). The extracellular NH2-terminal 220 residues have considerable sequence identity, including domains of nearly complete identity. For example, positions 94-161 (Fig. 1) have 25/68 residues identical between all polypeptides, including the cystine loop at positions 138-152, which is highly conserved. In contrast, several regions show greater sequence variability among the different subunit classes (shown in red color). For example, the twenty NH2-terminal residues have no common residues, the putative extracellular sequence 162-215 has only three residues in common to all polypeptides (leucine 187, glutamine 189, and glycine 207), and the putative cytoplasmic domain positions 307-391 has only two such residues (two ,

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lysines at 328 and 330 [sequence numbered according to rat a1]). The extracellular stretch at positions 162-215 shows variability in glycosylation sites, and presumably in ligand binding domains. In comparing the inferred amino acid sequences of different a-subtype polypeptides (in the rat), about 70% overall identity exists (88% counting conservative replacements). Residues that are identical in all a but different in other candidate subunit types are shown in black. Interestingly, there are 63 residues in this category. These include three stretches of sequence: positions 310-319 at the cytoplasmic exit of M3, 382-390 at the cytoplasmic entrance to M4, and 416-419 at the extracellular exit of M4. These domains may contribute to some a-specific structure and function. Amino acids coded in white are also identical in all a (all types); those in gray are identical in at least two different types of subunit candidate. In addition to the highly similar membrane-spanning regions, virtually complete identity exists in most of the NH2-terminal extracellular domain for all the a polypeptides. The slight differences in this region (indicated by the color red) may be sufficient to contribute to the different GABA and benzodiazepine ligand binding affinities seen between subtypes, since the ligand binding sites are probably in this stretch. Some small variations among a subtypes involve putative glycosylation sites. For example, two short stretches differ between a1 and a4, with a putative glycosylation site asparagine only in the a4, a fiveresidue stretch at positions 172-176 and another sevenresidue stretch at 198-204 (a1 sequence) (34). As mentioned for the a subtypes, the putative cytoplasmic loop between M3 and M4 has some similarity for all types in sequences near the membrane. All have several lysines in region 319-335. However, the major differences between candidate subunit types are primarily in this putative intracellular region: only about 20% identity is present for positions, 307-393, and a highly variable sequence is evident from positions 326-375, as indicated by the red amino acid residues in Fig. 1. This region would appear to contribute significantly to pharmacological subtype differences, as well as to variable cellular regulatory phenomena, and to differences between receptor classes. Figure 2 is a Venn diagram describing the number of identical amino acids between the published clones for rat GABAA receptor polypeptides: a1, a2, and a4 (32-34), i3, 132, and f3 (31), ‘12, and 6 (32). Interestingly, in addition to the 91/428 21% identity between all the cDNAs, the extent of identity among polypeptide types varies from 41 residues/428 10% (a and ‘y) and 34/428 8% (/3 and 6) to only 9/428 2% between a and 6; a-/3, /3-y, and y-& are intermediate at 17-21/428 4-5% (in addition to the 21% identity between all subunits). Thus the a and -y are more closely related than a and /3, a and 6, or -y and 6; /3 and 6 are more closely related than /3 and a, and ‘y, or -y and 6. The incidence of amino acid identities between three-subunit combinations of a/3-y (20/428), cryb (16/428), and /3-y#{244} (16/428 3.7-4.6%) are roughly

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=

=

=

=

=

/3,

=

OLSEN AND

TOBIN


The model in Fig. 3 does not indicate which polypeptide types constitute the subunits of a single receptor molecule. It is likely that each oligomer contains at least one copy of an a and /3 subunit, possibly more than one of each, and possibly other subunit types; this, however, remains to be established. The a type is sufficiently abundant and widespread that at least one copy of at least one a subtype is probably present in each and every oligomer. Although there are several /3 subtypes, available evidence does not allow a decision on whether one /3 subtype is always a constituent subunit. The -y type appears to provide essential properties to the oligomers (30). The ‘12 subtype is abundant, and its localization in the rat overlaps considerably with the a1 and /32 subunits (30, 32). Thus one type of a-y subunit may also be present in every oligomer. The 6 clone, however, appears to be localized completely differently from the ‘12 (and therefore also at least somewhat from the a1 and /32) (32); thus, some other a, and -y subunits must be associated with 6, or oligomers without all of these subunits exist. It is possible, for example, that either 6 or y is present in a given oligomer, and that those with 6 (32) correspond to subpopulations that have little or no sensitivity to benzodiazepines (42, 55, 56). Available evidence (such as the

/3,

Figure

2. Venn diagram summarizing amino acid sequence identity between the four types of polypeptide subunit candidates a, /3, y, and b of the rat GABAA receptor. The eight known sequences (see Fig. 1) were compared for pairwise, three-way, or all four subunittype matches at each residue. The regions of overlap are identified for the various squares in the diagram; the numbers indicate the number of identical amino acids of a total of 428 (for a,). Thus 91/428 = 21% are common to all the clones, 41 are common to a and y, an additional 20 are common the three types a, /3, and y, and 16 more are identical in the three types a, ‘y, t5, etc. Thus, the total number of identical amino acids between a and -y is 91 + 41 + 20 + 16 168/428 = 39%. Likewise, overall identities calculated for the other pairs are: a and /3(32%), a and b (29%), (3and -y (34%), (3and .5(34%), and ‘y and .5(33%).

equal in abundance, but identity within the a/36 trimer is rare (only 6/428 1.4% amino acid identity). This may be related to the low incidence of identity between a and 6 in pairwise comparison. Figure 3 is a model of the hetero-oligomeric GABAA receptor channel structure, based heavily on the wellstudied nicotinic receptor, another member of the =

ligand-gated

ion channel

receptor

superfamily

(5, 6, 9).

The number of subunits is not known, but is probably four or five, based on target size (27, 28), hydrodynamic measurements (17, 18), and analogy with the nicotinic receptor (7, 10, 52). Each subunit shows four membrane-spanning regions as in Fig. 1. One or more of these membrane-spanning regions of each subunit contributes to the wall of the ion channel. In the case of the nicotinic acetylcholine receptor, most evidence suggests that the M2 domain is involved (5, 8, 53, 54). By analogy, the M2 region is suggested here without evidence to play that structural role in the GABAA receptors. An invariant arginine residue is present at the extracellular exit of M2 in all subunits, and there is one arginine within M2 (but also in other membrane-spanning helices). The cationic amino acids could contribute to attracting anions into and through the channel.

variable

sequence

identities

described

in Fig. 2) sug-

gests that -y could substitute for a and form f3y oligomers, whereas 6 could substitute for 3 and form a#{244} oligomers. Despite the fact that the a,13,-y,6 nomenclature implies that each of these types must be present in each oligomeric subtype (true subunits), no one has enough evidence to reach any such conclusions. One or more of these subunits might substitute for another, rather than all being present simultaneously; such is the case in the nicotinic acetylcholine receptor, where #{128} replaces ‘1 during muscle fiber maturation (57). In any event, the subtype variation in pharmacological and physiological properties is due to the composition of the subunit subtypes included in the oligomer (or oligomers) of any given cell.

FUNCTIONAL RECEPTOR

EXPRESSION SUBUNIT GENES

OF

GABAA

The expression of proteins, including membrane receptors and channels, in heterologous cells injected with messenger RNAs allows one to assay the presence of appropriately coding mRNA and to study the properties of the encoded polypeptides (58, 59). The cloned bovine a1 plus /3 cDNAs were transcribed into sense RNA molecules and coinjected into Xenopus oocytes to produce GABA-dependent inward currents (presumably chloride channels) (2). A dose-dependent response to GABA (EC50 2 tM) was observed, with a reversal potential of 27 mV, and blockade by bicuculline and picrotoxin, plus potentiation by pentobarbital and the =

benzodiazepine

chlorazepate

(2).

Extrapolation from the oocyte expression system to the neuron in the brain might not be appropriate; the

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Plan View

heterologous cell expression must remain as only one of several factors necessary in determining the in vivo subunit composition. Further analysis of the a- and /3-derived channels revealed that most properties of the native GABAA receptor seen in neurons were present in the receptors produced from cloned cDNAs. However, two properties were lacking: 1) the cooperative sigmoidal doseresponse to GABA, and 2) the large stimulation by benzodiazepines (29, 46). Furthermore, mRNA for any individual polypeptide alone was capable of forming GABA-activated channels, with pentobarbital and picrotoxin modulation in oocytes (60) or cultured animal cells (15, 32). Although channels built from a single type of polypeptide had properties different from normal hetero-oligomeric channels, these experiments show that each polypeptide is structurally capable of forming channels, and that each contains functional binding sites for GABA, pentobarbital, and picrotoxin. Furthermore, each of the individual polypeptides produced homo-oligomeric channels with conductance substates detected by patch-clamp recordings (60). This indicates that multiple conductance states previously seen for GABAA responses in intact neurons (14) may be an intrinsic property of individual subunits rather than a result of receptor subtype heterogeneity. The sigmoidal dose-response to GABA has been partially reproduced in oocyte expression of cloned a and /3 subunit genes (35, 61). Apparently this property is technically difficult to demonstrate, rather than a missing property. Benzodiazepine modulation also can be seen with a/3 mixtures, but the response is either small (2, 29, 46) or inappropriate; one such mixture, for example, showed modest potentiation by inverse agonist as well as agonist ligands for the benzodiazepine site (35, 61). Channels expressed in cultured cells cotransfected with the cDNAs of a, /3, and ‘12 subunits showed a benzodiazepine response more similar to that of native receptors (30). These cells also expressed benzodiazepine receptor binding (30, 62). Receptors produced from any individual subunits, or a plus /3, or the a, /3, plus 6 cDNAs did not exhibit benzodiazepine binding (32, 62). The different a-subtype cDNAs show differential sensitivity to substrate GABA, physiological modulation, and pharmacological manipulation in heterologous cell expression (29, 34, 62). The three bovine a clones, expressed in Xenopus oocytes with /3, gave a halfmaximal response to GABA of about 1 tM for a2, 10 tM for a1, and more than 10 tM for a3 (29). In comparing the properties of channels expressed from the rat a1 plus /3 with those produced from a4 plus /3, the halfmaximal concentration of GABA was about the same (5 tM), but the maximal response to GABA was much bigger with a1. Both were inhibited by picrotoxin, but the a4 plus /3 channels (34, 43) or /3 alone (63) produced outward currents to picrotoxin in the absence of exogenous GABA, the apparent result of picrotoxin block of spontaneously opening channels produced by the GABA receptor mRNAs. Such picrotoxin responses have not been observed in neurons, but spontaneous

#{248}#{248}#{174}

Figure 3. Model of the GABAA receptor-chloride channel protein complex. The ligand-gated ion channel is proposed to be a heterooligomer composed of five subunits of the type shown in Fig. 1. Each subunit has four membrane-spanning domains (cylinders numbered 1-4), one or more of which contribute to the wall of the ion channel. The structure is patterned after the well-characterized nicotinic acetylcholine receptor, another membrane of the same gene superfamily (which has five subunits in electric organ but may have four in the brain [refs 5-8]), and is similar to that proposed by others for the GABAA receptor, except they suggest four subunits, which is equally likely (refs 2, 35). The naturally occurring oligomers are composed of some of thea, /3, ‘y, and .5 polypeptides, but the exact subunit composition, stoichiometry, and number of subunits are not known at this time.

oocyte may not be capable of proper processing of the gene product or of reproducing the mammalian cell membrane environment for the receptor. On the other hand, expression of an activity from certain mRNAs in oocytes or cultured animal cell lines suggests (but does not prove) that those mRNAs are necessary and sufficient for the native activity in a cell. Indeed, in the case of a multisubunit protein like the GABAA receptor, 1474

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TABLE

1. Comparison of rat a cDNAs

Number of amino Leader sequence Identity in amino with bovine: a, a2 a3 Messenger RNA Sizes (kb)

acids

428 27

421 30

433 31

99 76 70

70 96 70

70 71 66

acids

Distribution Hippocampus

Cerebral Cerebellum Striatum Substantia Olfactory

a4

a2

a,

Sequence

3.8

3

4.3

6 +

+

+ + +

+

+

-

-

+

cortex

+

+

+

±

nigra bulb

+

+

-

+

+

+

-

+

+

+

+

+

-

-

Thalamus Expression in oocytes GABA currents EC50(cM) Average maximal responses (to 50 jM GABA) Sensitive to picrotoxin Picrotoxin currents ANT, not tested.

2.8

±

BIOLOGY

VARIABLE GABAA

+

Nr

(nA) 80

284 + +

OF GABAA RECEPTORS

DISTRIBUTION

RECEPTOR

SUBUNITS

OF

MRNA AND

FOR SUBTYPES

+

5

5

+ +

+

openings of GABAA chloride channels can occur in neurons (64), and have been seen in oocytes injected with mRNA for a2 (60), i3 (61), and presumably other subunits and combinations. Table 1 summarizes the properties of the three rat a-subunit clones that we have described. Expression of a4 with both /3 and ‘1 in oocytes gave channels more sensitive to GABA than those produced from /3-y plus either a1 or a3 (63). Further, diversity in benzodiazepine pharmacology of the various a subunits, suspected from photoaffinity labeling studies (43, 65), can be reproduced by expression studies. Two pharmacological subtypes of benzodiazepine receptors have been defined by high (type I) or low affinity (type II) for the triazolopyridazine drugs such as CL 218,872 (42, 66, 67), and high (BZ1) or low affinity (BZ2) for /3-carbolines (68). Expression of the three a cDNAs (one at a time) in human 293 cells along with i3 and 72 resulted in detectable BZD binding to membrane homogenates. The binding showed type I/BZ1 specificity for the a1 subunit and type II/BZ2 properties for a2 or a3 (62). In addition, the a3-containing oligomers expressed a higher degree of GABA enhancement of benzodiazepine binding (about fourfold) compared with about twofold for a1 and a2 (62). Although it is not possible to extrapolate to the in vivo situation from these observations, differential properties of expressed subunit subtypes are likely to carry over to some extent. Multiple /3-subunit clones were expressed by coinjection with a cDNAs into Xenopus oocytes to demonstrate that they were authentic GABAA receptors, but pharmacological distinctions between different /3 subtypes

MOLECULAR

were not described (31). The /3 subtypes were considered less important than a in determination of benzodiazepine binding subtype characteristics (62). GABA ligand dose-response curves might be affected by the nature of the /3 subtype, since these appear to vary in GABA binding affinities (43, 44). Multiple -y subtypes also appear to exist, and they may have different pharmacological properties (D. Pritchett, personal communication). Future expression studies should produce much more information on structural, functional, and regulatory aspects of GABAA receptors. For example, sitedirected mutagenesis and domain-specific antibodies can be employed, along with protein chemistry, to identify important residues and domains within the polypeptide sequences.

Several groups have studied the distribution and development of GABAA receptor mRNAs using Northern blot analysis and in situ hybridization methods. The original bovine a1 and f3 clones were used to produce anti-sense oligonucleotide probes for mRNA detection in tissue sections by in situ hybridization. These showed mRNA for a1 present in all Purkinje and granule cells and some cells of the molecular layer of the bovine cerebellum, whereas mRNA for /3 was present only in the granule cell layer, not in Purkinje cells, and therefore only partially overlapped (69). The distribution of these two mRNAs in rat brain was investigated with the corresponding rat cRNA probes (70). Both mRNAs were present in somata in many regions corresponding to those showing a dense dendritic localization of GABAA/benzodiazepine receptor binding. Distribution of the a1 and /3 mRNAs overlapped but did not correspond exactly (70). The distribution of a1 mRNA in the rat was also studied by in situ hybridization with a human probe (71) that detected three distinct sizes of mRNA; the distribution revealed in these experiments was similar to that expected from GABAA receptor binding. A different human a1 probe was used to demonstrate that the levels of mRNA in rat brain increased during the first month after birth, as expected from benzodiazepine binding activity (72). The lack of total agreement between a1 and /3i distribution results from the existence of multiple a and /3 subtype genes and the presence in the brain of some oligomeric receptors that do not contain both a1 and /3 subunits. At least four different a clones have been described: bovine a1, a2, and a3 (29), rat a1 (33, 34, 35, 70), a2, and a4 (34), human a1 (30, 38, 71, 72), and mouse a1 (J. Sikela, personal communication). Three distinct /3 sequences have been described for cows and rats (31, 33) and possibly humans (30), at least two ‘ysubtypes from humans and rats (30, 32), and one 6 clone (32). The mRNAs for a subtypes have been extensively studied in cows and rats. In the cow, Northern blots

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showed that a1 mRNA was most abundant in the cerebellum and more abundant than the mRNAs of a2 and a3 in cortex, cerebellum, and hippocampus. In these three regions, a2 mRNA had the highest concentration in the hippocampus, whereas a3 mRNA was highest in the cortex (29). In situ hybridization showed the a1 mRNA to be abundant also in cells of the bovine inferior colliculus, olfactory bulb, and substantia nigra, whereas a2 and a3 were very low, but all three were present at relatively high concentrations in striatum (37). Differential localization of the three was seen in the cortex, with a1 and a2 mRNAs abundant in layers IT-TV, and a3 mRNA in layers V-VT. In the hippocampal formation, a2 mRNA was abundant in the dentate gyrus and CA3 region, whereas a1 was evenly distributed, and high in the subiculum and entorhinal cortex; a3 mRNA was present only at low levels (36). Comparing the rat a1, a2, and a4 mRNA distributions by in situ hybridization, we also found the a1 mRNA to be abundant and widely distributed, with very high levels in cerebellum and cortex, medium levels in the olfactory bulb (mitral cells) and inferior colliculus, and significant but lower levels in globus pallidus, thalamus, hippocampus, substantia nigra, and some pontine nuclei; the caudate was low, and spinal cord was negative (34, 39, 40, 43; A. J. MacLennan et al., unpublished results). The a2 mRNA was less abundant overall, with moderate levels only in hippocampal formation, especially the dentate gyrus, and some cranial nerve nuclei. The caudate contained low but significant levels, and there was a low concentration detected in cortex, olfactory bulb granule cells, olfactory tubercle, and amygdala. The a4 mRNA was seen only in hippocampus, cortex, olfactory bulb granule cells, and basal forebrain (39, 40, 43). These results are also included in Table 1. Examples of the comparison of a1 and a4 mRNA distribution in rat cortex, cerebellar cortex, and hippocampal formation are shown in Fig. 4 (34). The ai (A) was more abundant in all layers of the cortex (I) and in the cerebellum (II). In the hippocampus (III), a1 mRNA was present in some pyramidal cells of CA3 as well as nonpyramidal cells, whereas a4 was limited to pyramidal cells, but present in more, possibly all, pyramidal cells as well as in dentate granule cells. An important study of the distribution of and 6 polypeptide types in rat brain showed that these two subunit candidates may be part of distinct receptor oligomer subtypes (32). The -y type showed a distribution more typical of that expected of the benzodiazepine receptor from binding studies, and was similar to that of the a1 and /32 mRNAs, the more abundant species of these two polypeptide types. This included high levels in the neocortex (all layers), cerebellar Purkinje cells, olfactory bulb (mitral and tufted cell layers), hippocampal formation (all cells), and inferior colliculus, as well as medium levels in globus palhidus, thalamus, and substantia nigra. By contrast, the 6 mRNA was found in areas showing high-affinity muscimol binding without accompanying benzodiazepine binding (42,

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56): lower binding in cortex layer IV than in other layers; high levels in olfactory bulb internal granule layer, thalamus, and especially in the cerebellum granule cell layer. Very low to nil levels were seen in the olfactory bulb mitral cells, globus pallidus, hippocampus pyramidal cells, substantia nigra, and cerebellar Purkinje cells, traditional GABAA/benzodiazepine receptor areas (32). This supports the theory that the expression of GABAA receptor polypeptides (candidate subunits and subunit subtypes) in a tissue-specific manner determines the pharmacological properties of GABA synapses in a given tissue.

SUMMARY OF GABAA IN THE BRAIN

RECEPTOR

SUBTYPES

Available evidence suggests that subtypes of GABAA receptors exist and that they have distinctive distributions in the brain. The evidence includes observations of regional heterogeneity in various properties: pharmacological specificity (73, 74); ligand binding affinities measured both on homogenates (52, 66, 68) and tissue section autoradiography (42, 46, 67); allosteric interactions between binding sites on the GABAA/benzodiazepine receptor complex (55, 75); and physicochemical properties (65, 75, 76). Two subpopulations of benzodiazepine binding sites have been described, the types I and II (distinguished by CL 218,872 affinity) (66) or BZI-BZ2 (distinguished by /3-carboline affinity) (68). Photoaffinity labeling of benzodiazepine-binding polypeptides suggests three to five distinct gene products (65, 76). We have demonstrated in homogenate binding that type I/BZ1 and type II/BZ2 do not correspond completely, and that at least three subtypes are needed to explain GABA-benzodiazepine-barbiturate allosteric interactions in vitro; further, benzodiazepine binding sites show a regional variation in GABA coupling (sensitivity to in vitro modulation by GABA and bicuculline) that does not correspond to the type I/IT heterogeneity (55). In addition, the comparison of binding distribution in different rat brain regions by autoradiography has shown consistent discrepancies between both GABA and benzodiazepine ligands (56), but also between benzodiazepine ligands (67; J. G. Richards, personal communication), between GABA ligands (42), and between cage convulsants and GABA or benzodiazepine ligands (42). Comparison of the rat brain regional distribution for binding of seven ligands of the GABAA receptor complex revealed significant discrepancies between every pair. This was related to variable affinity of receptors for any given ligand; the data required at least four subtypes of receptor based on binding evidence alone (42). The four binding subtypes include a high-affinity muscimol binding site enriched in cerebellum granule cell layer and thalamus with little affinity for benzodiazepine ligands, a BZ1-like population enriched in substantia nigra, cerebellum molecular layer, and cortex layer IV, and a BZ2-hike population enriched in hip-

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Figure 4. In situ hybridization histochemistry of rat brain sections probed with 35S RNAs transcribed from a, (A) and a4 (B) cDNAs of rat GABAA receptor. The figures are dark field photographs. I) Cerebral cortex, with layers 1-VI numbered. Cells in all layers are labeled with both probes, with a, labeling more intense than a4. Calibration bar, 200 jzM. II) Cerebellum, with molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). No a4 transcript is detectable. Calibration bar, 100 tM. III) Hippocampal formation, with the dentate gyrus (DG) and field CA3 of Ammon’s horn (CA3). Some cells outside the pyramidal cell layer are heavily labeled with the a, probe; the a4 probe appears to label the vast majority of cells in both the granule and pyramidal cell layers. Calibration bar, 200 4M. Reproduced with permission from Khrestchatisky et al. (34).

superior colliculus, and cortex layers I-Ill, and a fourth population enriched in nucleus accumbens with high affinity for both bicuculline and SR95531, but not for muscimol (42). The BZ2 population appears to represent a variety of polypeptide clones, peptide bands on gels, and binding site properties, and this name should be dropped (although it could be redefined as non-BZ1). On the other hand, the BZ1 population can be correlated by several techniques with the a1 clone and 51-kDa peptide band photolabeled with [3H]flunitrazepam (34, 36, 37, 62, 65, 70, 76). Shivers et al. (32) have suggested that the ‘12 clone may correlate with benzodiazepine receptors showing low or relatively less GABA coupling (defined above), whereas the 6 clone may correlate with GABA receptors that show pocampus,

MOLECULAR

BIOLOGY

OF GABAA RECEPTORS

a high affinity for muscimol binding but low affinity or absent benzodiazepine binding (42). Clearly, there are more than two types of GABAA receptors, based on structural as well as functional diversity. Current studies are putting together the pieces of a jigsaw puzzle that will match the multiple clones with multiple polypeptide bands and multiple binding sites. This then can be related to the variable tissue location, physiological functions, and regulatory mechanisms for the family of GABAA receptors. The differences in a-subtype pharmacology expressed from clones is likely to be related, with some caution, to the situation in neurons. The evidence includes variable GABA dose-dependence (29, 63), maximal responses to GABA (34), and picrotoxin-induced currents (34, 63)

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for different a subtypes expressed in oocytes, as well as different benzodiazepine binding pharmacology for different a subtypes expressed in mammalian cells. Different degrees of GABA enhancement of benzodiazepine binding were also found with the expressed aclone subtypes (62). Tentatively, we can conclude that the different binding affinities for several ligands observed across rat brain regions by autoradiography (42) are related to these different subunit combinations, and that there are distinct pharmacological subtypes in the brain as a result. This conclusion is supported by the finding of polypeptide heterogeneity with different binding properties in purified GABAA receptor protein. Multiple polypeptide bands can be detected by protein staining on SDS gels (24, 43, 44, 52, 77) from purified GABAA receptor protein. These appear so far to correspond to multiple a and /3 subunits, as identified by photoaffinity labeling with benzodiazepine and GABA ligands, and by antibodies (43, 44, 78-82): either polyclonal antisera to the purified receptor (21) or monoclonal antibodies recognizing specific receptor subunits (25, 26). The different bands can be shown to produce distinct onedimensional peptide map fragments after proteolysis, indicating that they are distinct sequences (43, 44). This microheterogeneity of both a and /3 subunits also has pharmacological significance, since the various bands on SDS gels labeled with [3H]muscimol or 3H]fiunitrazepam have different affinities for nonradioactive analogs to inhibit binding. Sieghart and colleagues (65, 76) have shown that multiple polypeptides are labeled in crude brain homogenates with [3H]fiunitrazepam, and that these bands vary in affinity for some benzodiazepine ligands such as CL 218,872. We found in the purified receptor that the 58-kDa 3 subunit has a higher affinity for several GABA analogs than the 56-kDa 3 subunit (43, 44), and the 53-kDa a subunit gave more enhancement of benzodiazepine binding by GABA than the 51-kDa a band. The different polypeptides also varied in sensitivity to allosteric enhancers, such as barbiturates and steroid anesthetics (43, 44). The pharmacologically distinct subtypes defined by physically distinct bands on SDS gels show a brain regional variation, as expected from distinct gene products. Further, we found that the two 56- and 58-kDa /3 subunits showed differential activity as substrates for protein kinases C and A (50). Thus, protein chemistry and binding reveal pharmacologically and biochemically distinct receptor subtypes, consistent with the evidence from molecular cloning, in vitro expression, and in situ hybridization for receptor subtypes. A combination of these approaches is necessary to establish the subunit composition, localization, and physiological and pharmacological properties of the receptor populations that exist in any cell and brain region. Now that these multiple gene products for GABAA receptor polypeptides have been isolated, regulation of their expression in various interesting biological situa-

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tions, such as development, plasticity, diseases, and dependence on drugs such as alcohol and benzodiazepines, can be studied. Again, we have provided rat cDNAs to various groups that are asking important questions along these lines. In addition, the sequencing of the genomic DNA will be completed soon, and this will no doubt suggest mechanisms of regulation for expression for this interesting gene family. E!J We thank the following people for research described: Nicolas Brecha,

their contributions Michel Bureau,

Chiang, Meyer Jackson, Michel Khrestchatisky, MacLennan, Catia Sternini, and Wentao Xu.

Helen We thank

to the Ming-Yi Kim, John the follow-

ing people for helpful discussions: Michael Browning, Lynn Deng, Shuichi Endo, Carolyn R. Houser, J. Grayson Richards, Christian Ruppert, Geoff Smith, and James K. Wamsley. We thank Sharon Belkin for excellent illustrations. Supported by National Institutes of Health grants NS22071 (to R. W. Olsen); Tobin); and NS21908 (to A. V. Delgado-Escueta).

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NS22256

(to A.

J.

ES

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29. Levitan, E. S., Schofield, P. R., Burt, D. R., Rhee, L. M., Wisden, W., K#{246}hler, M., Fujita, N., Rodriguez, H. F., Stephenson, F. A., Darlison, M. G., Barnard, E. A., and Seeburg, P. H. (1988) Structural and functional basis for GABAA receptor heterogeneity. Nature (London) 335, 76-79 30. Pritchett, D., Sontheimer, H., Shivers, B. D., Ymer, S., Kettenmann, H., Schofield, P. R., and Seeburg, P. (1989) Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature (London) 338, 582-585 31. Ymer, S., Schofield, P. R., Draguhn, A., Werner, P., K#{246}hler, M., and Seeburg, P. H. (1989) GABAA receptor 3 subunit heterogeneity: functional expression of cloned cDNAs. EMBOJ 8, 1665-1670 32. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., K#{246}hler,M., Schofield, P. R., and Seeburg, P. H. (1989) Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron 3, 327-337 33. Lolait, S. J., O’Carroll, A.-M., Kusano, K., Muller, J. -M., Brownstein, M. J., and Mahan, L. C. (1989) Cloning and expression of a novel rat GABAA receptor. FEBS Lett. 246, 145-148 34. Khrestchatisky, M., MacLennan, A.J., Chiang, M.-Y., Xu, W., Jackson, M., Brecha, N., Sternini, C., Olsen, R. W., and Tobin, A. J. (1989) A novel alpha-subunit in rat brain GABAA receptors. Neuron 3, 745-753 35. M#{246}hler,H., Maiherbe, P., Draguhn, A., Sigel, E., Sequier, J. M., Persohn, E., and Richards, J. G. (1990) GABAA-receptor subunits: functional expression and gene localization. In GABA

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The FASEB journal OLSEN AND TOWN 1480 Vol. 4 March 1990 www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

Molecular biology of adrenergic receptors.

Molecular biology of dopamine receptors.

Molecular biology of serotonin receptors.

Molecular biology of serotonin receptors and transporters.

Molecular biology of the dopamine receptors.

Molecular biology of the glutamate receptors.

Molecular biology and respiratory disease. 5. Molecular biology of receptors: implications for lung disease.

The molecular biology of heparan sulfate fibroblast growth factor receptors.

Receptor mechanisms. Structure and molecular biology of transmitter receptors.

Dopamine directly modulates GABAA receptors.

Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes.

Functional sites for anesthetics in GABAA receptors.

Neurosteroids act on recombinant human GABAA receptors.

Biology of the TAM receptors.

Assessment of Methods for the Intracellular Blockade of GABAA Receptors.

Novel positive allosteric modulators of GABAA receptors with anesthetic activity.

Ethanol-induced plasticity of GABAA receptors in the basolateral amygdala.

Characterization of the picrotoxin site of GABAA receptors.

Metabolic Products of Linalool and Modulation of GABAA Receptors.

Molecular biology of cytomegalovirus.

Molecular biology of retroelements.

Molecular biology of papovaviruses.

Interneuronal GABAA receptors inside and outside of synapses.

Funding of molecular biology.