A propofol binding site on mammalian GABAA receptors identified by photolabeling.

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both ß3 homopentamers and α1ß3 heteropentamers. The binding site is located within the ß subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.

Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry.

Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 γ-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the α1 subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α1 subunit was observed in all three replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 γ-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the α1 subunit, confirmed two splice variants of the γ2 subunit (γ2L and γ2S) and resolved a database discrepancy in the sequence of the α5 subunit.

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells.

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ß3 subunit of the GABA(A) receptor.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5ß)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-ß3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-ß3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 µM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-ß3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ß3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3ß,5ß)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-ß3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ß3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies.

Idiopathic generalized epilepsies account for 30% of all epilepsies. Despite a predominant genetic aetiology, the genetic factors predisposing to idiopathic generalized epilepsies remain elusive. Studies of structural genomic variations have revealed a significant excess of recurrent microdeletions at 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 in various neuropsychiatric disorders including autism, intellectual disability and schizophrenia. Microdeletions at 15q13.3 have recently been shown to constitute a strong genetic risk factor for common idiopathic generalized epilepsy syndromes, implicating that other recurrent microdeletions may also be involved in epileptogenesis. This study aimed to investigate the impact of five microdeletions at the genomic hotspot regions 1q21.1, 15q11.2, 16p11.2, 16p13.11 and 22q11.2 on the genetic risk to common idiopathic generalized epilepsy syndromes. The candidate microdeletions were assessed by high-density single nucleotide polymorphism arrays in 1234 patients with idiopathic generalized epilepsy from North-western Europe and 3022 controls from the German population. Microdeletions were validated by quantitative polymerase chain reaction and their breakpoints refined by array comparative genomic hybridization. In total, 22 patients with idiopathic generalized epilepsy (1.8%) carried one of the five novel microdeletions compared with nine controls (0.3%) (odds ratio = 6.1; 95% confidence interval 2.8-13.2; chi(2) = 26.7; 1 degree of freedom; P = 2.4 x 10(-7)). Microdeletions were observed at 1q21.1 [Idiopathic generalized epilepsy (IGE)/control: 1/1], 15q11.2 (IGE/control: 12/6), 16p11.2 IGE/control: 1/0, 16p13.11 (IGE/control: 6/2) and 22q11.2 (IGE/control: 2/0). Significant associations with IGEs were found for the microdeletions at 15q11.2 (odds ratio = 4.9; 95% confidence interval 1.8-13.2; P = 4.2 x 10(-4)) and 16p13.11 (odds ratio = 7.4; 95% confidence interval 1.3-74.7; P = 0.009). Including nine patients with idiopathic generalized epilepsy in this cohort with known 15q13.3 microdeletions (IGE/control: 9/0), parental transmission could be examined in 14 families. While 10 microdeletions were inherited (seven maternal and three paternal transmissions), four microdeletions occurred de novo at 15q13.3 (n = 1), 16p13.11 (n = 2) and 22q11.2 (n = 1). Eight of the transmitting parents were clinically unaffected, suggesting that the microdeletion itself is not sufficient to cause the epilepsy phenotype. Although the microdeletions investigated are individually rare (<1%) in patients with idiopathic generalized epilepsy, they collectively seem to account for a significant fraction of the genetic variance in common idiopathic generalized epilepsy syndromes. The present results indicate an involvement of microdeletions at 15q11.2 and 16p13.11 in epileptogenesis and strengthen the evidence that recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 confer a pleiotropic susceptibility effect to a broad range of neuropsychiatric disorders.

No association of the neuropeptide Y (Leu7Pro) and ghrelin gene (Arg51Gln, Leu72Met, Gln90Leu) single nucleotide polymorphisms with eating disorders.

BACKGROUND: Genetic factors likely contribute to the biological vulnerability of eating disorders. AIMS: Case-control association study on one neuropeptide Y gene (Leu7Pro) polymorphism and three ghrelin gene (Arg51Gln, Leu72Met and Gln90Leu) polymorphisms. METHODS: 114 eating disorder patients (46 with anorexia nervosa, 30 with bulimia nervosa, 38 with binge eating disorder) and 164 healthy controls were genotyped. RESULTS: No differences were detected between patients and controls for any of the four polymorphisms in allele frequency and genotype distribution (P > 0.05). Allele frequencies and genotypes had no significant influence on body mass index (P > 0.05) in eating disorder patients. CONCLUSION: Positive findings of former case-control studies of associations between ghrelin gene polymorphisms and eating disorders could not be replicated. Neuropeptide Y gene polymorphisms have not been investigated in eating disorders before.

Additional support for linkage of schizophrenia and bipolar disorder to chromosome 3q29.

After publishing a genome scan and follow-up fine mapping, suggesting schizophrenia and bipolar disorder linkage to chromosome 3q29, we now genotyped 11 additional SNPs (single nucleotide polymorphisms), in order to narrow down a potential candidate region. Linkage was performed using the GENEHUNTER program version 2.1r3. A NPL score Z(all) of 3.891 (p=0.000156) was observed with SNP rs225. In short, we found significant linkage scores most telomeric on chromosome 3q29, spanning 3.46 Mbp (7 SNPs).

Formation of GABAA receptor complexes containing α1 and α5 subunits is paralleling a multiple T-maze learning task in mice.

There is limited information on the role of GABA type A receptors (GABAARs) containing α1, α5 and γ2 subunits in learning and memory. Here, we assessed the possible role of such receptors in spatial learning using the multiple T-maze (MTM) paradigm. C57BL/6J mice were trained in the MTM which induced elevated levels of α1 and α5 subunit-containing hippocampal GABAAR complexes. Moreover, spatial learning evoked a significant increase in the colocalization of α1 and α5 subunits in both, CA1 and dentate gyrus regions of the hippocampus suggesting the formation of complexes containing both subunits. Additionally, the presence of α1, α5 and γ2 subunits in high molecular weight GABAARs was detected and significant correlation in the level of α1-containing complexes with those containing α5 and γ2 subunits was demonstrated. Accordingly, α1 deficiency led to decreased levels of γ2 subunit-containing complexes, however, had no effect on α5-containing ones. On the other hand, α1 knockout mice showed impaired performance in the MTM correlating with increased levels of α5 subunit-containing GABAARs in comparison to trained floxed control animals which quickly learned the task. Taken together, these results suggest that α1, α5 and γ2-containing hippocampal GABAAR complexes play an essential role in spatial learning and memory in which targeted disruption of the α1 subunit produces profound deficits.

Genome scan for susceptibility loci for schizophrenia and bipolar disorder.

BACKGROUND: Despite the widely accepted view that schizophrenia and bipolar disorder represent independent illnesses and modes of inheritance, some data in the literature suggest that the diseases may share some genetic susceptibility. The objective of our analyses was to search for vulnerability loci for the two disorders. METHODS: A genomewide map of 388 microsatellite DNA markers was genotyped in five schizophrenia and three bipolar disorder Austrian families. Linkage analyses was used to compute the usual parametric logarithm of the likelihood of linkage (LOD) scores and nonparametric linkage analysis (NPL scores Z(all)) was used to assess the pattern of allele sharing at each marker locus relative to the presence of the disease (GENEHUNTER). Affected status was defined as severe affective disorder or schizophrenia. RESULTS: Across the genome, p values associated with NPL scores resulted in evidence (i.e., p <.0007) for linkage at marker D3S1265 on chromosome 3q (NPL score Z (all) = 3.74, p =.0003). Two other markers (on 3q and 6q) showed p values of <.01. CONCLUSIONS: We detected a potential susceptibility locus for bipolar disorder and schizophrenia on chromosome 3q, which has not been reported previously. The possibility of a false positive result has to be taken into account. Our data suggest shared loci for schizophrenia and bipolar affective disorders and are consistent with the continuum model of psychosis.

Synthesis, in vitro affinity, and efficacy of a bis 8-ethynyl-4H-imidazo[1,5a]- [1,4]benzodiazepine analogue, the first bivalent alpha5 subtype selective BzR/GABA(A) antagonist.

The synthesis and in vitro affinity of the alpha5beta3gamma2 (alpha5) subtype selective BzR/GABA(A) antagonist 7 is described. This ligand is selective for alpha5 subtypes in vitro and is a potent antagonist of the effects of diazepam only at alpha5beta3gamma2 subtypes (oocytes). Ligands such as 7 will be important in the determination of which physiological function(s) are subserved by this GABA(A) alpha5 subtype.

Possible linkage of schizophrenia and bipolar affective disorder to chromosome 3q29; a follow-up.

The present linkage study is a follow-up within the chromosome 3q29 region in schizophrenia and bipolar affective disorder families, based on our recently published genome scan, resulting in evidence for linkage of both disorders to this region (marker D3S1265: NPL [non parametric lod] score Z(all)=3.74, P=0.003). Using the same family sample (five pedigrees with schizophrenic index patients and three pedigrees with index bipolar disorder patients N=86; 50 of them were available for genotyping), genotyping of eight additional markers close to D3S1265 was done. Five of those new markers (three centromeric and two telomeric of D3S1265) spanning 4.14 cM (centiMorgan) could be used for statistical analyses ("new markers"). Moreover, marker D3S1265, genotyped within the published genome scan, was used for additional calculations. Linkage analysis was performed using the GENEHUNTER program version 2.1r3. Within newly genotyped markers the highest NPL score Z(all) observed was 1.93296 with the telomeric SNP (single nucleotide polymorphism) rs1835669, corresponding to P=0.032166. Statistical analysis including D3S1265, located in between the newly genotyped markers, resulted in a peak NPL score Z(all)=4.00179 with marker D3S1265, that is P=0.000128. Doing subset analyses of the bipolar disorder and schizophrenia families separately with new markers and D3S1265, linkage signals arose substantially from bipolar disorder families, with contribution from schizophrenia families, too. The results of our follow-up study support our previous linkage finding of schizophrenia and bipolar affective disorder to chromosome 3q29.

Histaminergic pharmacology of homo-oligomeric ß3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology.

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric ß3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to ß3 GABA(A) receptors with micromolar affinity (K(D)<300 µM), thus extending previous evidence that ß3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with ß3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric ß3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for ß3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether ß3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.

Bipolar disorder susceptibility region on chromosome 3q29 not confirmed in a case-control association study.

OBJECTIVES: We identified a bipolar disorder (BPD) susceptibility region on chromosome 3q29 in a genome-wide linkage scan (Bailer et al. 2002 (Biol Psychiatry 52: 40), NPL-score 4.09) and follow-up linkage analysis (Schosser et al. 2004 (J Psychiatr Res 38(3): 357), NPL-scores >3 with five markers). These findings were supported by further fine-mapping of this region (Schosser et al. 2007 (Eur Neuropsychopharmacol 17(6-7): 501)), finding NPL-scores >3.9 with SNPs (single nucleotide polymorphisms) spanning a region of 3.46 Mbp in BPD families. Since genetic association studies are more powerful than linkage studies for detecting susceptibility genes of small effect size, we aimed to replicate these findings in an independent case-control sample collected in London (UK) and Vienna (Austria). METHODS: A total of 51 SNPs were genotyped using Sequenom MassARRAY(®) iPLEX Gold and tested for association in a sample of 526 cases suffering from DSM-IV and/or ICD-10 diagnosis of BPD and 691 controls. RESULTS: No genotypic and/or allelic association, as well as no haplotypic association, was found for any SNP after multiple testing correction. CONCLUSIONS: However, we cannot exclude the possibility that our sample might not have the power to detect rare variants associated with susceptibility to BPD.

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians.

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains.

The analysis of highly hydrophobic proteins is still an analytical challenge. Using a recombinant gamma-aminobutyric acid A (GABAA)-receptor subunit as a model protein, we developed a gel-based proteomic approach for high MS/MS-peptide sequence coverage identification. Protein samples were separated by multi-dimensional gel electrophoresis and the three protein spots representing the GABAA-receptor subunit alpha-1 from the last electrophoretic step were used for in-gel digestion with trypsin, chymotrypsin and subtilisin, followed by subsequent mass-spectrometric identification by nano-ESI-LC-MS/MS Qstar XL (quadrupole time-of-flight (qQTOF)) and linear ion trap (LIT) LTQ XL identification. This protocol allows the unambiguous identification of the GABAA-receptor alpha-1 subunit protein with 100% sequence coverage, thus covering all four hydrophobic transmembrane domains. This protocol differs from other methods in the selection of enzymes, digestion conditions and use of the two mass spectrometry principles. The protocol takes approximately 10 d to complete and may represent a step forward in the complex analysis of other membrane or hydrophobic proteins.

15q13.3 microdeletions increase risk of idiopathic generalized epilepsy.

We identified 15q13.3 microdeletions encompassing the CHRNA7 gene in 12 of 1,223 individuals with idiopathic generalized epilepsy (IGE), which were not detected in 3,699 controls (joint P = 5.32 x 10(-8)). Most deletion carriers showed common IGE syndromes without other features previously associated with 15q13.3 microdeletions, such as intellectual disability, autism or schizophrenia. Our results indicate that 15q13.3 microdeletions constitute the most prevalent risk factor for common epilepsies identified to date.

Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits representing strongly hydrophobic transmembrane proteins.

GABA(A) receptors are the major inhibitory transmitter receptors in mammalian brain and are composed of several protein subunits that can belong to different subunit classes, leading to enormous heterogeneity. To establish techniques for the analysis of GABA(A) receptors in complex mixtures such as brain tissue, recombinant receptors composed of alpha1 and His-tagged beta3 subunits expressed in insect cells were purified by affinity chromatography and run on blue native gels. After denaturing, receptors were subjected to one- or two-dimensional electrophoresis in SDS-gels. Proteins were cleaved by multienzyme proteolysis and subjected to nano-ESI-LC-MS/MS. Both GABA(A) receptor subunits were well-separated and unambiguously identified by sequence coverage of 99.1% (alpha1) and 92.9% (beta3).

A GABRB3 promoter haplotype associated with childhood absence epilepsy impairs transcriptional activity.

Childhood absence epilepsy (CAE) is considered to exhibit a complex non-Mendelian pattern of inheritance. So far, only few CAE susceptibility genes have been identified. In a previous study of our group, an association between the GABA(A) receptor beta3 subunit (GABRB3) gene and CAE was shown. To further investigate this association, we screened 45 CAE patients of the first study for mutations in the 10 exons, the exon-intron boundaries and the regulatory sequences of GABRB3. Although we found no functionally relevant mutation, we did identify 13 single nucleotide polymorphisms (SNPs) in the GABRB3 gene region from the exon 1a promoter to the beginning of intron 3. Using these SNPs we defined four haplotypes for the respective GABRB3 gene region. A transmission disequilibrium test in the same 45 CAE patients and their parents indicated a significant association of this region and CAE (P=0.007075). Reporter gene assays in NT2 cells using exon 1a promoter constructs indicated that the disease-associated haplotype 2 promoter causes a significantly lower transcriptional activity than the haplotype 1 promoter that is over-represented in the controls. In silico analysis suggested that an exchange from T (haplotype 1) to C (haplotype 2) within this promoter impairs binding of the neuron-specific transcriptional activator N-Oct-3. Electrophoretic mobility shift assays demonstrated that the respective polymorphism reduces the nuclear protein binding affinity, thus explaining the results of the reporter gene assays. Reduced expression of the GABRB3 gene could therefore be one potential cause for the development of CAE, pathogenetically relevant in our patient group.

Investigation of the abundance and subunit composition of GABAA receptor subtypes in the cerebellum of alpha1-subunit-deficient mice.

In cerebellum, 75% of all GABAA receptors contain alpha1 subunits. Here, we investigated compensatory changes in GABAA receptor subunit expression and composition in alpha1 subunit-knockout mice. In these mice the total number of cerebellar GABAA receptors was reduced by 46%. Whereas the number of receptors containing alpha6 subunits was unchanged, the total amount of alpha6 subunits was significantly elevated. RT-PCR showed no increase of alpha6 mRNA levels, arguing against increased biosynthesis of these subunits. Elevated levels of alpha6 subunits in alpha1 -/- mice might thus have been caused by an increased incorporation of unassembled alpha6 subunits, replacing alpha1 subunits in alpha1alpha6betagamma2 or alpha1alpha6betadelta receptors, thus rescuing alpha6 subunits from degradation. Elevated levels of alpha3 and alpha4 subunits in the cerebellum of alpha1 -/- mice possibly can be explained similarly. Finally, a small amount of receptors containing no gamma or delta subunits was identified in these mice. Results suggest a total loss of GABAA receptors in cell types where alpha1 was the only alpha subunit expressed and a partial compensation for receptor loss in cell types containing other alpha subunits. Our results do not support a significant compensatory synthesis of other GABAA receptor subunits in the cerebellum of alpha1 -/- mice.

The 5'-flanking region of the rat GABA(A) receptor alpha2-subunit gene (Gabra2).

The GABA(A) receptor alpha2-subunit gene (Gabra2) has a specific spatial and temporal pattern of expression in rat brain. As a first step towards understanding the molecular mechanism underlying this regulation, we have investigated the structural properties of the 5'- flanking region of the rat Gabra2 gene. We identified six alpha2 transcript isoforms, each of which differs only in the 5'-untranslated region (UTR). Alignment of cDNA and genomic DNA sequences revealed that six 5'-UTRs are generated from three alternative first exons by alternative splicing using internal and terminal 5'-splice donor sites present in these exons. Promoter regions containing multiple transcription initiation sites were identified in the 5' proximity of each first exon. Two of these promoters lack TATA and CCAAT sequences. Finally, we have shown that differential activation of alternative promoters is used for the expression of the alpha2 mRNA isoforms during brain development, and that the diversity at the 5'-end of these transcripts affects GABA(A) receptor expression. Taken together, these results suggest that the expression of the Gabra2 gene can be influenced at both the transcriptional and post-transcriptional levels.