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The Journal of Neuroscience, May 15, 2000, 20(10):3588-3595

GABAA Receptor epsilon  and theta  Subunits Display Unusual Structural Variation between Species and Are Enriched in the Rat Locus Ceruleus

Saku T. Sinkkonen1, 2, Michael C. Hanna3, Ewen F. Kirkness3, and Esa R. Korpi1

1 Department of Pharmacology and Clinical Pharmacology, and 2 Turku Graduate School of Biomedical Sciences, University of Turku, FIN-20520 Turku, Finland, and 3 The Institute for Genomic Research, Rockville, Maryland 20850


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, GABAA receptor epsilon  and theta  subunits have been identified only in human. Here, we describe properties of the epsilon  and theta  subunit genes from mouse and rat that reveal an unusually high level of divergence from their human homologs. In addition to a low level of amino acid sequence conservation (~70%), the rodent epsilon  subunit cDNAs encode a unique Pro/Glx motif of ~400 residues within the N-terminal extracellular domain of the subunits. Transcripts of the rat epsilon  subunit were detected in brain and heart, whereas the mouse theta  subunit mRNA was detectable in brain, lung, and spleen by Northern blot analysis. In situ hybridization revealed a particularly strong signal for both subunit mRNAs in rat locus ceruleus in which expression was detectable from the first postnatal day. Lower levels of coexpression were also detected in other brainstem nuclei and in the hypothalamus. However, the expression pattern of theta  subunit mRNA was more widespread than that of epsilon  subunit, being found also in the cerebral cortex of rat pups. In contrast to primate brain, neither subunit was expressed in the hippocampus or substantia nigra. The results indicate that GABAA receptor epsilon  and theta  subunits are evolving at a much faster rate than other known GABAA receptor subunits and that their expression patterns and functional properties may differ significantly between species.

Key words: rat GABAA receptor subunits; subunit sequence variation; brain regional localization; locus ceruleus; hypothalamus; subunit coexpression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eighteen vertebrate GABAA receptor subunits have been categorized within seven families on the basis of sequence similarity (alpha 1-alpha 6, beta 1-beta 4, gamma 1-gamma 4, delta , epsilon , theta , and pi ) (Barnard et al., 1998; Bonnert et al., 1999). The beta 4 and gamma 4 subunits have been identified only in chick, whereas the most recently discovered subunits (epsilon  and theta ) have been described only in human. Notably, the human epsilon  and theta  subunits share greatest sequence similarity with the chick gamma 4 and beta 4 subunits, respectively (45-50% amino acid identity). Although the sequences of orthologous GABAA receptor subunits are generally well conserved between human and chick (83-98%), it is possible that the human epsilon  and theta  subunits are orthologs of the chick gamma 4 and beta 4 subunits, which have diverged to an unusually large extent. The proposal that the epsilon  and theta  subunit genes have mutated at a relatively rapid rate is supported by their chromosomal locations. The clustering of theta , alpha 3, and epsilon  subunit genes on human chromosome Xq28 (Levin et al., 1996; Wilke et al., 1997; Bonnert et al., 1999) indicates a common ancestry with the three beta /alpha /gamma subunit gene clusters in the human genome (Greger et al., 1995; McLean et al., 1995; Kostrzewa et al., 1996; Russek, 1999). However, whereas the alpha 3 subunit has retained strong similarity to other members of the alpha  subunit family, the theta  and epsilon  subunits have diverged significantly from their ancestral beta - and gamma -like subunit sequences.

If the human epsilon  and theta  subunits are orthologs of the chick gamma 4 and beta 4 subunits, respectively, the human genes have clearly diverged from their common ancestors to a much greater extent than the chick genes. This raises the possibility that the epsilon  and theta  subunits can confer properties to human GABAA receptors that are absent from chick. To better understand the divergence of epsilon  and theta  subunit orthologs and to examine their unique properties in different species, we have identified genes that encode homologs of epsilon  and theta  subunits in rodents. Recently, several reports have begun to describe expression patterns of rodent epsilon  and theta  subunits, using probes that were derived from homologous human subunits (Brooks-Kayal et al., 1998; Bonnert et al., 1999; Tobet et al., 1999). However, the rodent subunits described here display an unusually high level of sequence divergence from their human homologs. It is therefore essential to define the expression patterns of epsilon  and theta  subunits in rat brain using probes that are derived from rat cDNAs. Here, the brain regional expression patterns of epsilon  and theta  subunit mRNAs were studied by in situ hybridization (ISH) using rat subunit-specific oligonucleotide probes. The data suggest significantly different expression patterns for the subunits between rat and primate brain. Interestingly, a particularly strong enrichment of both subunits was observed in the locus ceruleus of adult and developing rats.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of rodent epsilon  subunit cDNAs. Primers were designed from exon 6 of the human epsilon  subunit gene (see below) for amplification of any homologous exons from rat genomic DNA. The primers were nucleotides 695-718 (sense) and 758-781 (antisense) of GenBank accession number U66661. Amplification at 95°C for 45 sec, 50°C for 60 sec, and 72°C for 2 min was performed for 30 cycles using the XL-PCR system (Perkin-Elmer, Norwalk, CT). A single reaction product was detected, purified from an agarose gel, and sequenced directly. Oligonucleotide primers were designed from the rat exon sequence to amplify the 5' and 3' flanking sequences from a rat brain cDNA library, using the Marathon system (Clontech, Palo Alto, CA). Amplification at 95°C for 45 sec, 60°C for 60 sec, and 72°C for 2 min was performed for 35 cycles using the XL-PCR system. Reaction products were purified from agarose gels and sequenced directly. Primers that flank the complete open reading frame of the rat subunit were then used to amplify a contiguous cDNA from the same library. The primers were 5'-tctagagtcgacGTCGTGCCAGGCAC-CGCTGAGATG and 5'-actagtctgcagGGTGATTGCCCCATGAGCTA-CCAG. Primers were also designed from the rat subunit cDNA sequence for amplification of homologous cDNA fragments from a mouse brain cDNA library (Clontech). Fragments of mouse cDNA that span the entire open reading frame of the homologous mouse subunit were purified from agarose gels and sequenced directly. Primers that flank the open reading frame were then used to amplify a contiguous cDNA from the same library. The primers were 5'-tctagagtcgacATGTTGCCTAAA-GTTCTCCTGATG and 5'-actagtctgcagCTGGAGCCTACAGGTTAA-GGCAAA. All cloned products were sequenced over their entire length to ensure that no mutations had been introduced.

Isolation of human and mouse epsilon  subunit genes. Two libraries of human genomic DNA, cloned in lambda DASH II and lambda FIX (Stratagene, La Jolla, CA), were screened at high stringency (Kirkness et al., 1991) with two 32P-labeled fragments of the human epsilon  subunit cDNA (nucleotides 20-328 and 695-1329 of GenBank accession number U66661). Sixteen hybridizing clones were obtained from ~2 × 106 plaques. Overlapping inserts were determined by restriction fragment mapping and Southern blotting. Exons and flanking introns were sequenced from templates of purified lambda  DNA using the Dye Terminator Cycle Sequencing system (PE Biosystems, Foster City, CA). The sequences of three genomic fragments that contain all of the exons have been deposited with GenBank under accession numbers U92281, U92282, and U92283. A library of mouse genomic DNA (strain 129SVJ), cloned in lambda  FIX II (Stratagene), was probed at moderate stringency [1× SSC (0.15 M NaCl and 0.0015 M Na-citrate) and 0.1% SDS, 50°C] with 32P-labeled fragments of the human and rat epsilon  subunit cDNAs (nucleotides 695-1329 of GenBank accession number U66661, and nucleotides 1-278 and 1611-3000 of GenBank accession number AF189262). Seventeen hybridizing clones were obtained from ~1 × 106 plaques. Overlapping inserts were determined by restriction fragment mapping and Southern blotting. Exons and flanking introns were sequenced from templates of purified lambda  DNA, and the sequences of five genomic fragments that contain all of the exons have been deposited with GenBank under accession numbers AF189264-AF189268.

Isolation of human, mouse, and rat theta  subunit cDNAs. The amino acid sequence of the chick GABAA receptor beta 4 subunit (GenBank accession number X56647) was used to search the nr and est databases of GenBank using the TBLASTN algorithm. Homologous peptide fragments were identified within the six-frame translations of GenBank accession numbers U47334 (a trapped exon from human chromosome Xq28) and W15780 (an expressed sequence tag from a fetal mouse cDNA library). Oligonucleotide primers were designed from these sequences to amplify 5' and 3' flanking sequences from human and mouse brain cDNA libraries using the Marathon system. Amplification at 95°C for 45 sec, 60°C for 60 sec, and 72°C for 2 min was performed for 35 cycles using the XL-PCR system. Reaction products were purified from agarose gels and sequenced directly. Sequences that flank the complete open reading frames of the human and mouse subunits were then used to amplify contiguous cDNAs from the same libraries. For the human theta  subunit cDNA (GenBank accession number AF189259), the primers contained nucleotides 1-24 (sense) and 1930-1953 (antisense). For the mouse theta  subunit cDNA (GenBank accession number AF189260), the primers contained nucleotides 11-34 (sense) and 1940-1963 (antisense). A fragment of the rat theta  subunit cDNA (GenBank accession number AF189261) was amplified from a rat brain cDNA library (Clontech) using primers derived from the mouse theta  subunit cDNA sequence. The primers were nucleotides 1394-1417 (sense) and 1940-1963 (antisense), and amplification at 95°C for 45 sec, 55°C for 60 sec, and 72°C for 2 min was performed for 35 cycles using the XL-PCR system. The reaction product was purified from an agarose gel and sequenced directly.

Northern blot analysis. Samples of ~2 µg of poly(A+) RNA (Clontech) were electrophoresed on a 1.2% formaldehyde agarose gel, transferred to nylon membranes, and hybridized with 32P-labeled fragments of the human theta  subunit cDNA (nucleotides 730-1953) or mouse theta  subunit cDNA (nucleotides 1602-1963). The blots were washed at 60°C in 0.1× SSC and 0.1% SDS before exposure. The blots were reprobed with 32P-labeled fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA (nucleotides 789-1140) (Tokunaga et al., 1987).

In situ hybridization. Male Sprague Dawley rats (n = 22; University of Turku, Turku, Finland) at four different ages [postnatal day 0 (P0), P6, P12, and adult] were killed, and brains were removed and frozen on dry ice. P0 rat heads were frozen as a whole. Fourteen micrometer thick sections were cut on a cryostat (Microm HM 500 OM; Microm Laborgeräte GMbH, Walldorf, Germany), mounted onto poly-L-lysine-coated slides, and dried at room temperature (RT) for 1-2 hr. Sections were fixed in 4% paraformaldehyde, washed in PBS for 5 min, dehydrated in 70% ethanol for 5 min, and stored in 95% ethanol at 4°C until used in hybridization.

ISH for detection of GABAA receptor subunit transcripts and localization of locus ceruleus was done using the protocol described by Wisden and Morris (1994). In detail, 36- to 45-bases-long antisense oligonucleotide probes were synthesized (Institute of Biotechnology, University of Helsinki, Helsinki, Finland) complementary to rat cDNA sequences. Tyrosine hydroxylase (TH) has been known to be heavily expressed in the locus ceruleus (Pickel et al., 1975), and a TH probe complementary to nucleotides 867-911 of rat cDNA (GenBank accession number M10244) was created to locate this nucleus in the rat brain. Rat GABAA receptor epsilon  subunit has been shown to have two different splice variants (Whiting et al., 1997). Three different probes were thus created to detect epsilon  subunit transcripts in the rat brain. They were complementary to (1) the boundary between exons 6 and 7 [functional variant (Whiting et al., 1997); nucleotides 2240-2275 of GenBank accession number AF189262], (2) exon 6 (both variants; nucleotides 2212-2256 of GenBank accession number AF189262), and (3) exon 9 (both variants; nucleotides 2616-2660 of GenBank accession number AF189262). Two probes against different positions [nucleotides 41-85 and 86-130 (probe 2; see Figs. 5-7) of GenBank accession number AF189261] of rat GABAA receptor theta  subunit mRNA were created to establish the expression pattern of this novel subunit.

Probes were [alpha -33P] (NEN, Boston, MA) 3' end-labeled with terminal transferase (Boehringer Mannheim, Mannheim, Germany) using 1:15-1:60 molar ratio of probe and radioactive nucleotide according to the labeling properties of different probes. Unincorporated nucleotides were separated by Bio-Spin 6 chromatography columns (Bio-Rad, Los Angeles, CA), and labeling efficiency was determined with a scintillation counter. One hundred microliters of hybridization buffer (50% formamide, 10% dextran sulfate, and 4× SSC) containing diluted probe (0.06 fmol/µl, 290-1100 dpm/µl) was applied to each slide and hybridized under parafilm coverslips overnight at 42°C. Sections were then washed in 1× SSC at RT for 10 min, 1× SSC at 55°C for 30 min, and finally through 3 min washing steps at RT as follows: 1× SSC, 0.1× SSC, 70% ethanol, and 95% ethanol. Sections were then air-dried and exposed to Biomax MR films (Eastman Kodak, Rochester, NY) with 14C standards for 3-12 weeks. Specificity of probes was determined with 100× excess of nonradioactively labeled probes. Images from representative films were produced by scanning the films using an HP ScanJet 4c/T scanner and HP DeskScan II program (Hewlett-Packard, Palo Alto, CA) and Adobe PhotoShop (version 3.0; Adobe Systems, Mountain View, CA). For anatomical localization, some of the serial slides were stained in 0.1% thionin (Sigma, St. Louis, MO) solution, washed in 70, 95, and 100% ethanol, air-dried, and mounted with Permount (Fisher, Pittsburgh, PA) and glass coverslips.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A fragment of the human epsilon  subunit cDNA (Davies et al., 1997) was used to isolate homologous cDNAs from rat and mouse brain (see Materials and Methods). The proteins that are encoded by these cDNAs are highly unusual (Fig. 1). Although they display all of the features that are characteristic of GABAA receptor subunits and are most similar to the human epsilon  subunit, they each contain a large insertion near the N terminus of the protein (Fig. 1B). This additional sequence (483 amino acid residues in the rat subunit) is composed primarily of Pro/Glu and Pro/Gln tandem repeats. However, in contrast to the peptide sequence, the encoding cDNA sequence contains few repetitive elements. It is therefore unlikely that this sequence has arisen from recent expansion of unstable repetitive units, as occurs in various genetic disorders (Reddy and Housman, 1997). The repetitive Pro/Glx element follows a predicted signal sequence (Nielsen et al., 1997) and is therefore expected to be located extracellularly.



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  		Figure 1.   Alignment of amino acid sequences for human, mouse, and rat GABAA receptor epsilon  subunit homologs. A, The translated human, mouse, and rat epsilon  subunit cDNAs were aligned after editing of the rodent sequences to remove a peptide fragment that is absent from the human sequence (see B). The normal location of this peptide fragment within the rodent sequences (after residue 50) is indicated (black-down-triangle ). Conserved residues are shaded, and the putative signal sequence (S) and transmembrane domains (M1-M4) are highlighted by lines above the corresponding sequences. Segments of the human and mouse subunits that are encoded by distinct exons are indicated by the locations of exon termini (up-arrow ). B, Alignment of the rodent-specific peptide fragments that were edited from A. Residues that are conserved between the rat and mouse sequences are shaded.

Notably, even if the repetitive Pro/Glx insertion is ignored, the rodent epsilon  subunits display only 68% amino acid identity with the human epsilon  subunit. This value is significantly less than that observed for rodent orthologs of other human GABAA receptor subunits (90-100%). This observation raised the possibility that the rodent subunits are a novel subtype of the epsilon  subunit class and that the true orthologs of the human epsilon  subunit had not been identified. However, screening of mouse genomic DNA libraries with the human epsilon  subunit cDNA at moderate stringency detected only the known mouse epsilon  subunit gene (see below). It was therefore concluded that there are no epsilon  subunit subtypes within the mouse genome that are more similar to the human epsilon  subunit than that which was identified by cDNA cloning. In common with previous Northern blot analyses of human epsilon  subunit mRNA (Garrett et al., 1997; Whiting et al., 1997; E. F. Kirkness, unpublished observations), transcripts of the rat epsilon  subunit (~7.2 kb) were relatively abundant in heart but were not detectable in samples of whole brain, liver, kidney, or skeletal muscle (data not shown).

It was of interest to determine whether the unusual sequence of the rodent cDNAs is derived from a distinct exon and whether such an exon also exists within the human epsilon  subunit gene. For this reason, the complete human and mouse epsilon  subunit genes were cloned and partially sequenced. The structure and sequences of the human epsilon  subunit gene (Fig. 2A) are essentially identical to those derived from an independent GenBank accession (number U82696). However, this structure conflicts with that reported by Wilke et al. (1997) in which intron 3 and a fragment of intron 6 were not identified. Their conclusion that the structure of the epsilon  subunit gene is different from all related subunit genes (Wilke et al., 1997) is not supported by this study. The structures of the human and mouse genes are identical (Fig. 2), and an additional exon cannot account for the unusual extra sequence of the mouse cDNA. It was conceivable that the repetitive Pro/Glx segments of the rodent subunits were derived from the use of an alternative 3' intron splice site between exons 1 and 2. However, the additional sequence of the mouse cDNA is not homologous to any region of intron 1 of the human gene. Furthermore, after PCR amplification of rat brain cDNA libraries with primers from exon 1 (sense) and exon 3 (antisense), all products were found to contain the complete exon 2 sequence. It is therefore concluded that the additional sequence of the rodent subunit genes is located within exon 2 and is not derived from a distinct exon or from the use of alternative splice sites.



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  		Figure 2.   Gene structures of human and mouse GABAA receptor epsilon  subunit homologs. A and B represent the human and mouse epsilon  subunit genes, respectively. For each panel, the top illustration represents the subunit mRNA. The protein-coding regions are shaded. Segments of the mRNA that are encoded by distinct exons are represented by numbered rectangles that are joined to the corresponding genomic sequence below. The bottom portion of each panel illustrates cloned fragments of genomic DNA.

Human and mouse theta  subunit cDNAs were first identified by searching GenBank for sequences that are homologous to the chick GABAA receptor beta 4 subunit. The identified sequence fragments were used to clone longer cDNAs from human and mouse brain cDNA libraries (see Materials and Methods). These cDNAs contain the complete open reading frames of the human and mouse theta  subunits (Fig. 3). The mouse cDNA sequence was also used to isolate a fragment of the rat theta  subunit cDNA for in situ hybridization studies (see below). The human theta  subunit cDNA encodes a polypeptide of 632 amino acid residues that is most closely related to the chick beta 4 subunit (56% amino acid identity in extracellular and transmembrane domains). After the completion of this work, Bonnert et al. (1999) reported a human cDNA sequence that is essentially identical to that described in Fig. 3. However, the cDNA described here encodes five additional amino acid residues at the N terminus of the subunit. The first Met codon is flanked by a consensus sequence for initiation of translation (Kozak, 1991), which is absent from that proposed previously (Met6; Bonnert et al., 1999). The only other difference between the two sequences is at Ile478 at which the sequence of Bonnert et al. (1999) encodes a Phe residue. The human and mouse theta  subunits share only 76% amino acid identity. Consequently, in common with the epsilon  subunit homologs, there was concern that the human and mouse cDNAs are not orthologous but represent distinct subtypes of a theta  subunit family. However, Southern blot analysis of mouse genomic DNA with the human theta  subunit cDNA detected only the known mouse theta  subunit gene (data not shown). Northern blot analysis of theta  subunit mRNA expression in human and mouse tissues indicated predominant expression in the brain (Fig. 4). Transcripts of the theta  subunit are relatively large, with human brain displaying at least two mRNA species of 7.3 and 8.0 kb. In mouse, a single transcript of 7.6 kb was detectable in brain, spleen, and lung.



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  		Figure 3.   Alignment of amino acid sequences for human, mouse, and rat GABAA receptor theta  subunit homologs. The amino acid sequences of theta  subunit homologs from human (full-length), mouse (full-length), and rat (partial) were translated from cloned cDNAs. Conserved residues are shaded, and the putative signal sequence (S) and transmembrane domains (M1-M4) are highlighted by lines above the corresponding sequences.



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  		Figure 4.   Distribution of GABAA receptor theta  subunit mRNA in human and mouse tissues. A, Poly(A+) RNA from human tissues (lanes 1-8) or mouse tissues (lanes 9-16) was hybridized with 32P-labeled fragments of the human and mouse theta  subunit cDNA, respectively. The mRNA was derived from heart (lane 1), whole brain (2), placenta (3), lung (4), liver (5), skeletal muscle (6), kidney (7), pancreas (8), heart (9), whole brain (10), spleen (11), lung (12), liver (13), skeletal muscle (14), kidney (15), and testes (16). B, The same blots were reprobed with a 32P-labeled fragment of the human GAPD cDNA.

Brain regional distribution of epsilon  and theta  subunit transcripts revealed a common site of strong expression in the adult rat, i.e., locus ceruleus in the brainstem (Fig. 5). The epsilon  subunit mRNA, visualized with three different oligonucleotide probes with identical results, was also lightly expressed in the hypothalamus but not in the cerebral cortex or hippocampus (Fig. 6). Signals from all probes were consistent with the expression of the long, functionally active splice variant of epsilon  subunit mRNA (Whiting et al., 1997). The theta  subunit mRNA, visualized with two different probes with identical results, was also detectable in the hypothalamus and some thalamic nuclei and was nonexistent in the hippocampus and cerebral cortex (Fig. 6). No significant amount of epsilon  and theta  subunit mRNA was found in the substantia nigra or caudate putamen (data not shown).



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  		Figure 5.   Localization of GABAA receptor epsilon  and theta  subunit mRNAs in the rat locus ceruleus. The subunits were localized by in situ hybridization using 33P-labeled specific oligonucleotide probes. Images from serial frontal sections are shown. LC, Thionin staining of the section probed with tyrosine hydroxylase; locus ceruleus in the box. TH, Tyrosine hydroxylase probe. theta , theta  subunit probe 2. epsilon 67e, epsilon  subunit probe against exon border 6-7. epsilon 6e, epsilon  subunit probe against exon 6. epsilon 9e, epsilon  subunit probe against exon 9. Hundred-fold excess of cold probes abolished the hybridization to the background level in each case, indicating specificity of the signals (data not shown).



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  		Figure 6.   Localization of GABAA receptor epsilon  and theta  subunit mRNAs in the adult rat hypothalamus. Images by in situ hybridization of epsilon  and theta  subunit transcripts were done using 33P-labeled specific oligonucleotide probes epsilon 67e and probe 2, respectively. Control, Thionin staining; CM, centromedial thalamic nucleus; Ctx, cerebral cortex; DMH, dorsomedial hypothalamus; Hi, hippocampus; Th, thalamus; VMH, ventromedial hypothalamus.

During postnatal development of the rat brain, both epsilon  and theta  subunit transcripts were detectable at birth in the locus ceruleus (Fig. 7). There was also weaker expression in the brainstem, including the dorsal raphe nuclei, for both subunits. The expression of theta  subunit mRNA appeared slightly more widespread, being also detectable in the inner layer of the cerebral cortex, especially at the postnatal day 6, and in the basal forebrain nuclei, such as the bed nucleus of stria terminalis. No clear signals for either epsilon  and theta  subunit transcripts were detected in the hippocampal regions, basal ganglia, and most of the cerebral cortex at postnatal days 0-12.



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  		Figure 7.   Expression of GABAA receptor epsilon  and theta  subunit mRNAs in the rat brain during postnatal development. Serial horizontal sections for in situ hybridization of epsilon  (probe epsilon 67e) and theta  (probe 2) are shown. Control, Thionin staining; LC, locus ceruleus; Ctx, cerebral cortex; DR, raphe dorsalis; BST, bed nucleus of stria terminalis; Hi, hippocampus.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cloning of rodent epsilon  and theta  subunit cDNAs has revealed an unusually large degree of sequence divergence from their human orthologs. Rodent epsilon  subunits also exhibit an unusual insertion of repetitive amino acid sequence within the large extracellular domain. There are few examples of such long repetitive Pro/Glx motifs in the databases of known protein sequences. Among eukaryotic genes, these include a microtubule-associated protein (Goedert et al., 1996), a putative transmembrane transporter (Lafreniere et al., 1994), and a repressor of apoptosis (Koseki et al., 1998). The function of the Pro/Glx motifs in these proteins is uncertain. For the putative transmembrane transporter, the Pro/Glu repeat has been classified as a PEST domain (Lafreniere et al., 1994), predicting a role in targeting the protein for rapid proteolysis (Rechsteiner and Rogers, 1996). To date, we have been unable to demonstrate unique functional properties that can be attributed to the rat epsilon  subunit when expressed with known GABAA receptor subunits in human embryonic kidney 293 cells. It is possible that the nascent polypeptide must undergo post-translational processing before assembly within functional receptors, and this will be pursued by probing native and recombinant subunits with subunit-specific antibodies.

For both the epsilon  and theta  subunits, the sequences of rodent and human homologs suggest that these subunits are evolving at a much faster rate than other known GABAA receptor subunits. Differences in the rates of mutation have been noted previously for other gene families. For example, the genes encoding alpha -fetoprotein and serum albumin likely arose from a gene duplication event (Kioussis et al., 1981) but have mutated at significantly different rates since the divergence of rodents and human (Minghetti et al., 1985). The biological function that is acquired by each new gene family member appears to impose unique structural constraints. This explanation suggests that the structural requirements for function of epsilon  and theta  subunits are more flexible than for other known GABAA receptor subunits. Consequently, they have evolved under less constraint and can tolerate more mutations. It is possible that their biological functions can be maintained by fewer conserved residues or that their functions in rodents and human have diverged. The large insertion within the rodent epsilon  subunits is clearly suggestive of at least some distinctive properties, and it will be important to define how the structural variation might distinguish neuronal activity in rodents and human.

Owing to the sequence variation between rodent and human epsilon  subunits, many probes for the human subunit are unlikely to cross-react with the appropriate rodent subunit. An antiserum that recognizes a peptide of the human subunit (residues 409-427) (Whiting et al., 1997) has been used for immunolocalization (Tobet et al., 1999) and immunoprecipitation (Bonnert et al., 1999) of rodent epsilon  subunits. However, the human peptide shares only ~30% sequence identity with either the rat or mouse epsilon  subunit peptides described here, and the data derived from the use of this antiserum in rodents are therefore questionable. In addition, a fragment of the human theta  subunit has been used to generate an antiserum for immunoprecipitation of rodent theta  subunits (Bonnert et al., 1999), although this peptide shares only 63% amino acid identity with the mouse theta  subunit described here.

The localized expression of these subunits is consistent with the fact that all GABAA receptor subunit genes have distinct expression patterns in the brain (Wisden et al., 1992). However, there is evidence that some clustered genes exhibit colocalized expression, such as the beta 2, alpha 1, and gamma 2 subunit genes on mouse chromosome 11 that very likely form the major GABAA receptor subtype in the brain (McKernan and Whiting, 1996). Our data support this idea by demonstrating that the clustered theta , alpha 3, and epsilon  subunit genes also show colocalized expression. Regions in which the epsilon  and theta  subunits are coexpressed are known to contain alpha 3 subunit mRNA (Wisden et al., 1992). Although the alpha 3 subunit is also expressed in other brain regions, it is predicted to assemble with epsilon  and theta  subunits in discrete brain regions, such as the locus ceruleus.

Our localization data differ from previously reported patterns for epsilon  and theta  subunit expression in primate brain (Whiting et al., 1997; Bonnert et al., 1999). There are clear differences in the hippocampus and substantia nigra, which show expression of epsilon  and theta  subunits in the monkey but not in the rat. Furthermore, the theta  subunit peptide has been reported to be strongly expressed in the rat striatum (Bonnert et al., 1999). This is inconsistent with the lack of detectable mRNA expression by in situ hybridization in the caudate putamen or substantia nigra pars compacta. Therefore, the existence of a native alpha 2beta 1gamma 1theta subunit combination, suggested to be responsible for ~20% of [3H]muscimol binding in the rat striatum (Bonnert et al., 1999), should be reexamined for the reasons concerning species specificity of the antisera (see above).

The most interesting localization for both epsilon  and theta  subunits was the bilateral noradrenergic locus ceruleus, which has been implicated with a number of behavioral and physiological processes, e.g., anxiety, panic, attention, and drug withdrawal syndromes. This nucleus has been "lacking" a normal repertoire of GABAA receptor subunits to form functional GABAA receptors (Luque et al., 1994a). The absence of gamma 2 subunit expression (Luque et al., 1994b) is likely to explain why benzodiazepines are often inefficient in reducing evoked neuronal activity in the locus ceruleus (Simson and Weiss, 1989; Beck and Loh, 1990). It is suggested that alpha 3beta theta epsilon receptors form a receptor population with unique locus ceruleus-enriched expression pattern and benzodiazepine insensitivity, which might be exploited to find subtype-selective nonbenzodiazepine site compounds for the treatment of stress and anxiety. The theta  subunit has been localized in the human locus ceruleus by immunohistochemistry (Bonnert et al., 1999), but no information is available for the epsilon  subunit. Furthermore, double or triple immunohistochemical technique should be used to directly demonstrate the colocalization of epsilon  and theta  subunits with alpha  and beta  subunits (Fritschy et al., 1992; Waldvogel et al., 1999). Because both epsilon  and theta  subunit mRNA was expressed already at birth, epsilon  and theta  subunit-containing receptors may be involved in selective trophic action of GABA on monoaminergic neurons (Liu et al., 1997).

In summary, our results provide the rational basis for examining the possible pharmacological modulation of discrete brain nuclei in the brainstem and hypothalamus by targeting epsilon  and theta  subunit-containing GABAA receptors. Furthermore, the low sequence conservation and distinctive expression patterns of these X chromosome-clustered genes indicate that their functional properties may differ significantly between human and rodents.


    FOOTNOTES

Received Jan. 3, 2000; revised March 9, 2000; accepted March 10, 2000.

This work was supported by National Institutes of Health Grant R29 NS34702 (E.F.K.) and the Academy of Finland (E.R.K.).

Correspondence should be addressed to Dr. Esa R. Korpi, Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520 Turku, Finland. E-mail: esa.korpi{at}utu.fi.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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