QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (188) Citing Articles via Google Scholar Google Scholar Articles by Jones, A. Articles by Wisden, W. Search for Related Content PubMed PubMed Citation Articles by Jones, A. Articles by Wisden, W.

 Previous Article  |  Next Article 

Volume 17, Number 4, Issue of February 15, 1997 pp. 1350-1362
Copyright ©1997 Society for Neuroscience

Ligand-Gated Ion Channel Subunit Partnerships: GABA_A Receptor ₆ Subunit Gene Inactivation Inhibits  Subunit Expression

A. Jones^{1, a}, E. R. Korpi^{2, a}, R. M. McKernan^{3, a}, R. Pelz^{4, a}, Z. Nusser⁵, R. Mäkelä², J. R. Mellor¹, S. Pollard⁶, S. Bahn¹, F. A. Stephenson⁶, A. D. Randall¹, W. Sieghart⁴, P. Somogyi⁵, A. J. H. Smith¹, and W. Wisden¹

¹ Medical Research Council Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge CB2 2QH, United Kingdom, ² Department of Pharmacology and Clinical Pharmacology, University of Turku, Fin-20014 Turku, Finland, Department of Alcohol Research, National Public Health Institute, Fin-00180 Helsinki, Finland, ³ Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, CM20 2QR, United Kingdom, ⁴ University Clinic for Psychiatry, Department of Biochemical Psychiatry, 1090 Vienna, Austria, ⁵ Medical Research Council Anatomical Neuropharmacology Unit, Oxford University Department of Pharmacology, Oxford, OX1 3TH, United Kingdom, and ⁶ School of Pharmacy, University of London, Brunswick Square, London, WC1N 1AX, United Kingdom

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

---

ABSTRACT

Cerebellar granule cells express six GABA_A receptor subunits abundantly (₁, ₆, ₂, ₃, ₂, and ) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact ₆ protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse ₆ subunit gene was disrupted by homologous recombination. In ₆ / granule cells, the  subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with  subunit-specific antibodies. The  subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the  subunit. These results provide genetic evidence for a specific association between the ₆ and  subunits. Because in ₆ / neurons the remaining ₁, _2/3, and ₂ subunits cannot rescue the  subunit, certain potential subunit combinations may not be found in wild-type cells.

Key words: GABA_A receptor; ₆ subunit; granule cell; cerebellum; homologous recombination; gene targeting; transgenic mice; knockout mice; ligand-gated ion channel; subunit sorting; subunit assembly; internal ribosome entry site; dicistronic mRNA; muscimol; SR95531; Ro 15-4513; flunitrazepam

---

INTRODUCTION

In vertebrate brains, GABA_A receptors are the principal mediators of inhibitory synaptic transmission. They are agonist-gated anion channels formed of pentameric assemblies of subunits arranged around an aqueous pore (Seeburg et al., 1990; Sieghart, 1995; Stephenson, 1995; Tyndale et al., 1995; McKernan and Whiting, 1996). The subunit genes (_1-6, _1-3, _1-3, and ) are differentially transcribed, and the polypeptides are assembled in many possible combinations depending on cell type (Persohn et al., 1992; Wisden et al., 1992; Fritschy and Möhler, 1995). There are several important unresolved issues. Why is this receptor heterogeneity needed for synaptic function? What is the subunit composition of native subtypes of receptor? Are there rules guiding which subunits assemble with each other?

The cerebellum is an excellent brain area for investigating these questions. Its clearly defined circuitry allows an almost complete account of which cerebellar cell types express which GABA_A receptor subunit genes (Wisden et al., 1996). For example, cerebellar granule cells express six subunit genes abundantly (₁, ₆, ₂, ₃, ₂, and ), and so they probably have several distinct GABA_A receptor subtypes of unknown subunit stoichiometry. As for the canonical muscle nicotinic acetylcholine receptor subunits (Verrall and Hall, 1992; Green and Claudio, 1993; Kreienkamp et al., 1995), there is likely to be selective discrimination between GABA_A subunits; however, assembling a neuronal receptor requires solving an extensive combinatorial element. To form a native receptor, subunits have to recognize and distinguish their neighbors. The assembly pathways used by granule cells to sort the six principal subunits into different receptor subtypes are not known. Granule cells receive a single GABAergic input from Golgi interneurons onto their distal dendrites. At this synapse, the GABA_A receptor subtypes might be colocalized and intermingled (Nusser et al., 1995, 1996), and to date the ₁, ₆, _2/3, and ₂ subunits have been demonstrated to be present in the synaptic junction (Nusser et al., 1995, 1996; Somogyi et al., 1996).

Despite the comparative simplicity of the system, the receptor subunit composition of granule cell GABA_A receptors is controversial. Current views accommodate _12/32, _62/32, _162/32, _162/32, _12/32, and _62/3 combinations (Korpi and Lüddens, 1993; Mertens et al., 1993; Caruncho and Costa, 1994; Khan et al., 1994, 1996; Quirk et al., 1994; Caruncho et al., 1995; Korpi et al., 1995; Pollard et al., 1995). The evidence for such combinations is derived from antibody-based data and correlation of pharmacological fingerprints of native binding sites, with binding profiles of subunits expressed in cell lines. Here we have targeted the ₆ subunit gene by homologous recombination techniques. Removal of ₆ protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied and provided genetic evidence for a specific association between the ₆ and  subunits. Our results begin to reveal the rules guiding receptor subunit assembly.

---

MATERIALS AND METHODS

Generation of mutant mice The replacement vector for homologous recombination, designed for positive-negative selection (Mansour et al., 1988), was generated from a 6 kb mouse 129 strain ₆ subunit gene fragment (Jones et al., 1996), comprising part of exon 4 through to the middle of intron 8. This fragment was subcloned into pBluescript (Stratagene, La Jolla, CA) (see Fig. 1A). Into this plasmid, a BamHI cassette (TAG₃IRESlacZpAMC1neopA) (Nehls et al., 1996) was inserted between the AflII and NcoI sites located in exon 8, after the site ends were modified by adding BamHI adaptors. This cassette, designed to report target gene expression and to provide a dominant marker to select for insertion into the gene, consisted of stop codons (TAG) in all three frames, followed by an internal ribosome entry site (IRES) linked to a lacZ reading frame and SV40 polyadenylation sequences. It also contained a neomycin resistance gene under independent transcriptional control (Nehls et al., 1996). The IRES-lacZ cassette was inserted with the lacZ coding sequence in the same transcriptional orientation as the ₆ gene coding sequences. For negative selection, a XhoI-SalI fragment containing two MC1tk gene head-to-tail repeats (Smith et al., 1995) was placed in the targeting vector polylinker appending the longer homology arm (see Fig. 1A). The vector was linearized with SalI and electroporated into 129 strain-derived embryonic stem (ES) cells (ES line "CCB," kindly supplied by Drs. W. Colledge and M. Evans, Wellcome/CRC, University of Cambridge).

---

Fig. 1. GABA_A receptor ₆ subunit gene disruption by homologous recombination. A, Wild-type ₆ gene, targeting (replacement) vector, and the disrupted ₆ gene structures. Numbers indicate exons. On the Replacement vector, the broken line indicates pBluescript (pBS) sequences. On the wild-type allele, the asterisk marks exon 8 where the IRES lacZ/neo cassette was inserted. Only relevant restriction sites are shown. A, AflII; B, BamHI; N, NcoI; S, SphI; X, XhoI. Arrows mark the neo and tk gene promoter sites and direction of transcription. The lacZ coding sequence orientation is the same as the ₆ gene, thus permitting its translation from the IRES sequence (striped box) to be initiated on the mRNA derived from the ₆ promoter. Expected restriction fragment lengths diagnostic for homologous recombination and the probes used to detect these are marked by double-headed arrows and horizontal bars, respectively. B, Confirmation of ₆ mutant allele germline transmission. Biopsy tail DNA samples were digested with SphI, electrophoresed, and Southern-blotted. The membrane was probed with PROBE A (3 flanking). Wild-type (+/+) individuals give a 15 kb band, the homozygous null (/) animals give a 9 kb band, and heterozygotes (+/) give both bands.
[View Larger Version of this Image (60K GIF file)]

---

Transfected ES cells were grown on G418^r primary embryonic fibroblast feeder cells, in medium supplemented with leukemia inhibitory factor (Life Technologies, Paisley, UK) and selected in G418 (Life Technologies) and FIAU (Bristol-Myers, Hounslow, UK) (Mansour et al., 1988; Smith et al., 1995). Genomic DNA was isolated from individual colonies, digested with SphI, Southern-blotted, and probed with an intron 8-derived SacI-XbaI restriction fragment (see PROBE A in Fig. 1A). The addition of the neomycin gene creates an additional SphI site in the ₆ gene locus, thus enabling the discrimination between wild-type (15 kb) and null (9 kb) alleles (see Fig. 1A,B). Confirmation of correct targeting events was established with restriction fragment probes from the lacZ and neo genes (marked on Fig. 1A) (data not shown) and probe B (the complete sequence of probe B, which comprises the promoter and 5 untranslated region, is deposited in the EMBL database, accession number X97475[GenBank]; Jones et al., 1996) (see Fig. 1A). Additional diagnostic restriction digests of targeted genomic DNA used BamHI (see Fig. 1A).

Male chimeras derived from targeted cells were mated with wild-type C57BL/6. Mutation germ-line transmission was determined by Southern blot analysis of agouti progeny tail DNA (see Fig. 1B). Heterozygotes were intercrossed to generate a homozygous ₆ / line.

-Galactosidase staining Mice were transcardially perfused with 4% paraformaldehyde (PFA) in PBS. Brains were removed, post-fixed for 1 hr in 4% PFA, and then equilibrated at 4°C in PBS containing 30% sucrose. Sections (40 µM) were cut on a sliding microtome and incubated free-floating in 5-bromo-4-chloro-3-indolyl--galactoside (X-Gal) (Bonnerot and Nicolas, 1993). After X-Gal staining, some sections were counterstained with neutral red (Sigma, Poole, UK), allowing non-lacZ-expressing cells to be visualized. Alternatively, whole brains (see Fig. 3C) were immersed in the X-Gal staining reagent.

---

Fig. 3. LacZ expression driven from the ₆ gene locus in ₆ / adult mouse brains illustrated in horizontal (A), coronal (B), and whole-mount views (C); blue coloration indicates lacZ activity. A and B show the confined expression of the ₆ gene to the cerebellar granule cell layer; C shows the expression in the dorsal regions of the inferior colliculi; D shows higher-power view of ₆ gene expression in the molecular layer of the cerebellum. The arrow indicates an example of the numerous lacZ positive cells in the molecular layer. These are probably nonmigrated granule cells. The arrowheads mark putative parallel fiber staining; E, ₆ gene expression in the dorsal cochlear nucleus granule cells. BS, Brainstem; Cb, cerebellum; CbGr, cerebellar granule cell layer; Ctx, neocortex; Gr, cerebellar granule cells; DCGr, dorsal cochlear nucleus granule cells; H, hippocampus; IC, inferior colliculi; Mol, cerebellar molecular layer. Scale bars: A, 1.3 mm; B, C, 1 mm; D, 150 µm; E, 300 µm.
[View Larger Version of this Image (116K GIF file)]

---

Staining of cultured granule cells. Cells on coverslips (see Granule Cell Culture and Electrophysiological Analysis) were washed in PBS and fixed in ice-cold 2% PFA/0.2% glutaraldehyde in PBS for 5 min. The coverslips were washed in PBS, incubated with X-Gal solution at 37°C overnight, and counterstained with neutral red.

Antibodies

₁-specific antibodies. ₁(1-9), an N-terminal-specific antibody (Zezula and Sieghart, 1991); ₁-N, affinity-purified, raised against rat N terminus residues 1-14 (S. Pollard and F. A. Stephenson, unpublished data). ₁(328-382)/₁L was prepared as described (Mossier et al., 1994). Rabbits were immunized with an MBP-₁(328-382)-7His fusion protein, and the antibodies were purified with a GST-₁(328-382)-7His fusion protein. This antibody precipitates GABA_A receptors and is selective for the ₁ subunit (R. Pelz and W. Sieghart, unpublished data).

₆-specific antibodies. ₆-N (Batch R54XV), affinity-purified polyclonal, was raised to bovine ₆ subunit N-terminal residues 1-16 (Thompson et al., 1992); ₆(429-434) batch P24, affinity-purified rabbit polyclonal antibody, was raised to rat ₆ subunit residues 429-434 (Tögel et al., 1994); ₆-C, affinity-purified rabbit polyclonal, was directed against the C-terminus sequence CSKDTMEVSSTVE (S. Pollard and F. A. Stephenson, unpublished data).

-specific antibodies. (318-400), rabbit polyclonal was raised against the rat cytoplasmic loop sequence between TM3 and TM4 (Quirk et al., 1995); (1-44) (rabbit R7) polyclonal was prepared by immunizing with an MBP-(1-44)-7His fusion protein and purifying by affinity chromatography, as described (Mossier et al., 1994; R. Pelz and W. Sieghart, unpublished data). This antibody is specific for the  subunit and does not precipitate 132 receptors (R. Pelz and W. Sieghart, unpublished data).

Immunocytochemistry Five ₆ / and five +/+ mice were transcardially perfused with 4% PFA, 0.05% glutaraldehyde, and ~0.2% picric acid for 7-17 min. After perfusion the brains were washed in 0.1 M phosphate buffer. Preembedding immunocytochemistry was carried out on 70-µm-thick vibratome sections (Somogyi et al., 1989). Floating sections were incubated in 20% normal goat serum (NGS) diluted in Tris-buffered saline (TBS), pH 7.4, for 1 hr. The purified antibodies were diluted in TBS containing 1% NGS. After they were washed, the sections were incubated for 2 hr in biotinylated goat anti-rabbit IgG (diluted 1:50 in 1% NGS containing TBS), followed by incubation in avidin-biotinylated horseradish peroxidase complex (diluted 1:100; Vector Laboratories, Peterborough, UK) for 90 min. Peroxidase enzyme reaction was with 3,3-diaminobenzidine tetrahydrochloride as chromogen and H₂O₂ as oxidant. In some cases, Triton X-100 (0.1-0.3%) was added to the TBS throughout the experiment. The antibody concentrations used for immunocytochemistry were (1-44)R7, 0.7-2.2 µg/ml; ₆-N, 1.5-3.0 µg/ml.

For controls, selective labeling could not be detected when the primary antibodies were either omitted or replaced by 5% normal rabbit serum. No immunoreactivity was obtained when the antibodies were preincubated with the appropriate peptides used for immunization (Nusser et al., 1996).

Ligand autoradiography The procedures were slightly modified from Olsen et al. (1990) and Wong et al. (1996). Cryostat sections (14 µm) from frozen nonfixed adult mouse brains were preincubated in 50 mM Tris-HCl, pH 7.4, and 120 mM NaCl for 15 min at 0°C, except for the GABA site assays when 0.31 M Tris-citrate solution, pH 7.1, was used. Incubations with ligands used fresh buffers of composition identical to those used for preincubation. For the benzodiazepine (BZ) site, [³H]Ro 15-4513 (5 nM, Du Pont de Nemours, NEN Division, Dreieich, Germany) was used with and without 100 µM diazepam (Orion, Espoo, Finland) for a 60 min incubation at 0°C, followed by three 30 sec washes, a dip in distilled water, and rapid drying. The same conditions and washes were used for the GABA site, with [³H]muscimol (20 nM, Amersham, Buckinghamshire, UK) and [³H]SR 95531 (20 nM, Du Pont), except that the incubation time was 30 min. The sections were washed three times for 15 sec in 10 mM Tris-HCl, pH 7.4, followed by dipping in distilled water and air drying. Sections were exposed to Hyperfilm-³H (Amersham) for 1-6 weeks. The images were produced by scanning the films. The nonspecific binding components to BZ and GABA sites were defined in the presence of 10 µM Ro 15-1788 (Hoffmann-La Roche, Basel, Switzerland) and 100 µM GABA, respectively.

Radioligand binding Radioligand binding on membranes prepared from individual mouse cerebella was as described previously (Quirk et al., 1994). Membranes prepared from each animal were used for saturation binding with [³H]Ro 15-1788 (0.1-17.0 nM), [³H]zolpidem (1-30 nM), and [³H]Ro 15-4513 (0.8-60.0 nM) displaced with Ro 15-4513 (10 µM) to define the total number of BZ binding sites, or with flunitrazepam (1 µM) to define binding to diazepam-sensitive sites only. Saturation binding with [³H]muscimol (2-45 nM) used 1 mM GABA to determine nonspecific levels. All assays used eight concentrations of ligand, with total and nonspecific binding measured in duplicate with 30-80 µg of protein/assay tube. B_max and K_d values were determined by nonlinear least-squares fit of the saturation curves using the data analysis software RS1 (Bolt, Beranek and Newman, Cambridge MA).

Immunoprecipitation analysis Immunoprecipitation of GABA_A receptors solubilized from individual mouse cerebella used antibody (318-400) bound to protein A-Sepharose as described previously (Quirk et al., 1994, 1995). [³H]muscimol binding to the solubilized receptor was measured after gel filtration through Sephadex G-25 to remove any remaining endogenous GABA.

PAGE and immunoblotting Membranes from individual +/+ and / cerebella were prepared, and equal amounts of protein per slot were subjected to SDS-PAGE in 10% polyacrylamide gels and immunoblotted. For the ₁(1-14), ₆(1-16), and ₆-C antibodies, the ECL Western blotting system (Amersham) was used for detection (Pollard et al., 1995). ECL blots were quantitated by normalizing with an anti-neuron specific enolase (NSE) antibody (Sigma) and then probing with ₁(1-14) (S. Pollard and F. A. Stephenson, unpublished data). Multiple exposures were taken for both anti-NSE and ₁(1-14) immunoreactivity and quantitated using a Molecular Dynamics Personal Quantitator. For the (1-44)R7 and ₁(328-382) antibodies, membranes were incubated with digoxygenin-labeled antibodies and were then treated with anti-digoxygenin-alkaline phosphatase Fab fragments (Boehringer Mannheim, Mannheim, Germany). Proteins were detected by fluorescence using the CSPD substrate (Tropix). Blots were quantitated by densitometry of Kodak X-Omat S films with the DocuGel 2000i gel documentation system using the RFLPscan software (MWG-biotech).

Granule cell culture and electrophysiological analysis Cell culture. Cerebellar granule cells, attached to matrigel-coated coverslips, were cultured from postnatal day 5 (P5) mice as described for rat cells (Randall and Tsien, 1995). Minimal essential medium was supplemented with glucose (5 mg/ml), transferrin (100 µg/ml), insulin (5 mg/ml), glutamine (0.3 mg/ml), and 10% fetal calf serum. After 2 d, the cells were fed with media that was supplemented further with 4 µM cytosine arabinoside, and they were then fed every 5 d by a 50% replacement of the culture media. Electrophysiological measurements were made after 14-17 d in vitro.

Electrophysiology Recordings were from single, visually identified, granule cells using both outside-out patches and whole cells pulled away from the underlying cell-attachment substrate. No differences were observed between data from patches and whole cells, and results from both data sets were therefore pooled. A piezoelectrically driven theta tube-based application system delivered 120 msec pulses of GABA. Concentration jumps from control to agonist and vice versa took place within ~1 msec. Five 120 msec 20 µM GABA pulses were applied at 0.1 Hz before and during the application of 1 µM flunitrazepam (Sigma). Recovery from the actions of flunitrazepam were studied with 20 additional GABA applications. Data were filtered at 2 kHz and sampled directly to computer at 10 kHz under control of the pClamp software suite. Because of the presence of some application-to-application variability in the current peak amplitude generated by GABA, an arbitrary threshold was set, with a 15% increase in the GABA response considered to be a potentiation above the baseline variability.

In situ hybridization In situ hybridization with ³⁵S-labeled oligonucleotide probes was as described (Wisden and Morris, 1994). The oligonucleotide sequences used were ₄: 5-TTCTGGACAGAAACCATCTTCGCCACATGCCATACTTTAAGCCTGT-3 (EMBL accession number L08493[GenBank]) and : 5-AGCAGCTGAGAGGGAGAAAAGGACGATGGCGTTCCTCACATCCAT-3 (EMBL accession number M60596[GenBank])

Behavioral observations The animals (n = 23 for both +/+ and / lines, from two generations) were observed in their normal activities. Open field explorative activity was determined, under artificial lighting, in a round area (diameter 83 cm) divided into 19 segments. The mice were in the area for the first time. Their behavior was recorded for 5 min with a video recorder, and the behavioral parameters (number of segment crossings with all four feet and number of rearings) were scored blindly afterward. The number of fecal boli was counted before the area was cleaned for the next animal. The ability of the mice to learn to climb up onto a thin horizontal wire while initially hanging from their forepaws was tested in three trials during 1 d. Their ability to learn to stay on an accelerating rotating rod (Rotamex, Columbus Instruments, Columbus, OH) for 180 sec was tested during daily sessions. The initial session was 3 min on a nonmoving rod. On subsequent sessions, the mice were placed on the stationary rod, and the rotation speed was then set at 5 rpm and increased to 15 rpm over a 180 sec interval.

---

RESULTS

Creation of a mouse line with no GABA_A receptor ₆ subunit protein: mapping ₆ expression with a dicistronic RNA encoding lacZ

Homologous recombination in embryonic stem cells was used to create a 129/Sv × C57BL/6 mouse line in which the ₆ subunit gene was disrupted at exon 8 (Fig. 1A,B) (see Materials and Methods). The mutation, located just after the TM2 (channel lining) region, consisted of an insertion of stop codons in all three reading frames, an IRES linked to a -galactosidase (lacZ) open reading frame and SV40 polyadenylation site, and finally a neomycin resistance gene expressed from its own promoter (Mountford et al., 1994; Nehls et al., 1996) (Fig. 1A). Translation of the ₆ subunit mRNA from the mutant allele should terminate just after the TM2 region, resulting in a 300 amino acid protein designated ₆M2. The stop codon-IRES insertion generates a dicistronic mRNA in which -galactosidase protein translation is then linked to an ₆ gene expression, i.e., lacZ expression is under ₆ transcriptional regulatory element control.

Homozygous mutant mice had no overt defects and could breed normally (see Behavioral Characterization of ₆ / Mice). By the criteria of Nissl staining, the size, folding of folia, and histological appearance of the cerebellum in ₆ / animals were completely normal. Immunocytochemistry with the ₆ N-terminal-specific antibody ₆-N (Thompson et al., 1992), however, demonstrated a total loss of ₆-specific immunoreactivity from the cerebellar granule cell layer of / mice (Fig. 2A,B). Using both N- and C-terminal ₆ subunit-specific antibodies (Thompson et al., 1992; S. Pollard and F. A. Stephenson, unpublished data), a complete loss of the 57 kDa immunoreactive ₆ band was also seen on Western blots of cerebellar protein extracts isolated from / animals (Fig. 2C). Identical results were found with an additional C-terminal anti-peptide antibody ₆(429-434) (R. Pelz and W. Sieghart, data not shown). Long exposure times of blots probed with the N-terminal antibody failed to show any ₆-specific degradation products in the / samples (data not shown), indicating that ₆M2 is not a stable entity. In a ₂ subunit gene knockout study, which similarly used an exon 8 disruption, a truncated ₂ form was also not detectable (Günther et al., 1995).

---

Fig. 2. Immunodetection of the ₆ subunit of the GABA_A receptor in ₆ +/+ (A, C) or ₆ / (B, C) cerebella as visualized with either light microscopic immunoperoxidase reactions (A, B) or immunoblotting (C). A, B, An intense immunoreactivity for the ₆ subunits in the granule cell layer (gr) disappeared in ₆ / mice (B). The sections and one immunoblot were reacted with the same N-terminal domain-specific antibody. Scale bars: A, B, 500 µm. C, The 57 kDa ₆ protein is absent in ₆ / cerebella, as shown with either ₆-N, an N-terminal-specific antibody, or ₆-C, a C-terminal-specific antibody to the ₆ subunit.
[View Larger Version of this Image (52K GIF file)]

---

We mapped ₆ gene expression in adult / animals using -galactosidase staining (Fig. 3). An intense blue coloration was seen in the cerebellar granule cell layer (Fig. 3A,B). The reaction product started to appear within the first 5 min of incubating the sections at room temperature in X-Gal and was fully developed within 30 min, thus demonstrating the extremely high level of ₆ locus expression. There were numerous small blue cells in the cerebellar molecular layer. These are probably ectopic granule cells (Fig. 3D) (cf. Thompson et al., 1992; Gao and Fritschy, 1995; Gutiérrez et al., 1996). Strong, blue coloration was also seen along the molecular layer outer edge, probably corresponding to -galactosidase enzyme transported into granule cell axons, the parallel fibers. As expected, there were many blue granule cells in the dorsal cochlear nuclei (Fig. 3E) (Varecka et al., 1994). With little exception, the rest of the brain showed no detectable staining (Fig. 3A). Unexpectedly, however, many cells in the inferior colliculi dorsal regions stained blue (Fig. 3C), and there were other minor cell populations in the substantia nigra and thalamus (geniculate nuclei) with faint but consistent blue staining (data not shown). These populations of stained cells were not seen in wild-type animals, and thus were not attributable to endogenous -galactosidase-like activity.

₆ subunit expression has not been noted previously in the inferior colliculi, substantia nigra, or thalamus by in situ hybridization or immunoreactivity, although a rat ₆ gene proximal promoter fragment consistently drives lacZ expression in the inferior colliculi of transgenic mice (Jones et al., 1996). The reasons for the lack of detection in previous studies could include low ₆ mRNA and protein levels. Alternatively, ₆ may be part of presynaptic receptors transported to distant axon terminals outside the inferior colliculi and so may escape detection in the inferior colliculi nucleus itself. The long half-life of -galactosidase in mammalian tissue contributes to the extreme sensitivity of the lacZ reporter method. Over time, low levels of transcription from the ₆-lacZ hybrid gene will lead to accumulating amounts of -galactosidase. These results make clear the usefulness of tracking gene expression using dicistronic-based reporters (Mountford et al., 1994; Nehls et al., 1996).

Pharmacological characterization of ₆ / cerebellar granule cells: BZ sensitivity

The ₆ protein absence was established further by pharmacological analysis. GABA_A receptors containing the ₆ subunit are insensitive to most types of BZs, such as diazepam or flunitrazepam (Lüddens et al., 1990; Hadingham et al., 1996). Ro 15-4513, however, is a BZ that binds to all subtypes of GABA_A receptor with _x2 subunit combinations, including those containing the ₆ subunit (Lüddens et al., 1990; Sieghart, 1995). Thus, a diagnostic assay for ₆ in cerebellar granule cells is the high level of [³H]Ro 15-4513 binding on granule cell membranes that is insensitive to full BZ agonists such as diazepam (Sieghart et al., 1987; Malminiemi and Korpi, 1989; Lüddens et al., 1990; Turner et al., 1991). Normally, more than half of the [³H]Ro 15-4513 binding in the granule cell layer is diazepam insensitive (DIS) but can be displaced by micromolar concentrations of the BZ antagonist flumazenil (also known as Ro 15-1788). This profile is thought to be attributable to the abundant expression of _62/32 receptors on granule cells (Lüddens et al., 1990; Korpi and Lüddens, 1993; Korpi et al., 1993; for review, see Wisden et al., 1996). In the ₆ / animals, DIS binding over the cerebellar granule cell layer is completely absent (Fig. 4A), whereas in wild-type brains [³H]Ro 15-4513 still binds over the granule cell layer even in the presence of 100 µM diazepam (Fig. 4A). From binding studies using isolated cerebellar membranes, the contribution that the ₆ subunit makes to the number of total cerebellar Ro 15-4513 binding sites was estimated to be ~40% (see Evaluation of ₁ Subunit Levels; also see Table 1).

---

Fig. 4. Autoradiographic analysis of GABA_A receptor binding sites in wild-type (+/+) and ₆ / mice. A, Benzodiazepine sites labeled by 5 nM [³H]R0 15-4513 showing total and diazepam-insensitive binding in the presence of 100 µM diazepam. B, GABA_A receptor sites labeled by 20 nM [³H]muscimol, showing total binding. The nonspecific signal in the presence of 100 µM GABA was at the film background level. C, GABA_A receptor sites labeled by 20 nM [³H]SR 95531, showing total binding. The nonspecific binding signal in the presence of 100 µM GABA was similar in wild-type and / brains (data not shown). Similar distinct pharmacological profiles were observed between the wild-type and ₆ / brains in each of seven pairs of adult mice studied. D, E, In situ hybridization x-ray film autoradiographs of adult mouse brains hybridized with ₄ (D) and -specific (E) ³⁵S-labeled oligonucleotide probes. Wild-type (+/+) brains are on the left; ₆ / brains are on the right. No differences can be seen in subunit mRNA levels between +/+ and / brains. Note also the very similar pattern of ₄ and  gene expression in the forebrain/thalamus regions, and the correlation with the distribution of [³H]muscimol (B). Cbgr, Cerebellar granule cells; CP, caudate-putamen; Ctx, cerebral cortex; Gr, cerebellar granule cell layer; H, hippocampus; IC, inferior colliculus; OB, olfactory bulb; T, thalamus.
[View Larger Version of this Image (86K GIF file)]

---

Table 1. Determination of Bz binding in 6 / mice Ligand +/+  / Kd Bmax Kd Bmax Ro 15-1788 0.91  ± 0.16 1222  ± 144 0.84  ± 0.17 1021  ± 141 Ro 15-4513 total sites 9.4  ± 2.0 2106  ± 203 6.3  ± 1.5* 1159  ± 107** DS 5.9  ± 0.8 1199  ± 114 5.7  ± 0.5 1088  ± 174 Zolpidem 17.9  ± 4 1155  ± 152 19.6  ± 4.8 993  ± 161 Muscimol 6.3  ± 0.8 2568  ± 326 6.2  ± 1.7 661  ± 234 Data shown are the mean ± SEM of cerebellar membranes prepared independently from six animals. Saturation analysis used eight concentrations of ligand in duplicate for each animal. Bmax (fmol/mg protein) and Kd (nM) values were determined by nonlinear least-squares fit of the saturation curves using the data analysis software RS1. * Significantly different from +/+ (p < 0.05); ** significantly different from +/+ (p < 0.005). DS, Diazepam sensitive.

The ₆ subunit has a closely related homolog, the ₄ subunit, which is expressed in certain forebrain areas such as the thalamus (Wisden et al., 1991, 1992). The recombinant ₄ subunit in an _4x2 configuration displays a pharmacological profile identical to that of _6x2 receptors, and ₄ mRNA is found at low levels in cerebellar granule cells of adult rats (Wisden et al., 1991; Laurie et al., 1992). Thus we looked to see whether there had been a compensatory change in ₄ expression in the cerebellum of ₆ / mice (Fig. 4D); however, consistent with the absence of DIS binding in ₆ / animals, there was no upregulation of ₄ mRNA in / cerebella (Fig. 4D).

The BZ sensitivity of GABA_A receptors in ₆ / cerebellar granule cells was investigated directly using electrophysiology on cultured granule cells isolated from P5 animals. After 14-17 d in vitro, we tested the effects of BZ agonist flunitrazepam (1 µM) coapplication on the current amplitude generated by 20 µM GABA. Results of a typical culture are shown in Figure 5. Examination of wild-type cells revealed a heterogeneous response: flunitrazepam-induced potentiation of the GABA response took place in approximately half (20 of 36) of the cells tested (Fig. 5C, top row). In those cells with flunitrazepam-potentiated GABA responses, the average potentiation was 58 ± 7%. In contrast, in age-matched cultures derived from ₆ / cerebella, 16 of 18 cells tested had flunitrazepam-induced potentiations of their GABA responses. The average potentiation was 62 ± 7% (Fig. 5C).

---

Fig. 5. Electrophysiological characterization of GABA_A receptors in cerebellar granule cells from wild-type and ₆ / cells. Photomicrographs in A and B show typical examples of lacZ-expressing cerebellar granule cells, isolated from P5 ₆ / mouse cerebella, and cultured for 3 weeks. A is a low-magnification view, showing the mosaic of blue (lacZ-positive) cells scattered throughout the culture. Both isolated and clustered blue cells can be seen. Within any given cluster, not all the cells are blue and therefore are not expressing the ₆ gene. The cells have been counterstained with neutral red. All electrophysiological recordings were from isolated cells. Arrows in B show examples of non-lacZ-expressing cells. Scale bars: A, 200 µm; B, 30 µm. C, Example responses to 120 msec applications of 20 µM GABA alone, and 20 µM GABA with 1 µM flunitrazepam (open arrows). The top row shows an example of wild-type cells (+/+) with GABA_A receptors that responded to flunitrazepam (left trace) or were insensitive to flunitrazepam (right trace). The bottom row shows a typical GABA_A response from an ₆ / cell and the associated flunitrazepam potentiation. From left to right and top to bottom, the value x on the scale bar corresponds to 200, 230, and 170 pA, respectively. The traces were averages of three to five consecutive records.
[View Larger Version of this Image (76K GIF file)]

---

To examine a possible reason for the heterogeneity of the GABA_A receptor response, the extent of gene expression from the ₆ locus in cultured ₆ /granule cells was analyzed with -galactosidase histochemistry. At 3 weeks in culture, numerous cells strongly stained dark blue after incubation with X-Gal (Fig. 5A,B); however, there were many adjacent "granule-like" cells that either contained just a few blue particles or were completely unstained (Fig. 5B). This applied both to isolated cells and to cells in large clusters. There was no obvious correlation between the location of cells (isolated or in clusters) and lacZ expression. This mosaic of blue cells is evidence that at least in culture, not all granule cells or granule-like cells express the ₆ gene (cf. Santi et al., 1994), and may explain the heterogeneous nature of the BZ potentiation seen in our cultures.

Selective  subunit protein loss from cerebellar granule cells of ₆ / mice

A key and controversial question for cerebellar granule cell GABA_A receptors has been which subunits co-assemble in vivo (Wisden et al., 1996). To examine one aspect of this, we immunoprecipitated deoxycholate-solubilized cerebellar GABA_A receptors from ₆ / mice with a -specific polyclonal antiserum, (318-400), raised against the putative intracellular loop domain between TM3 and TM4 (Quirk et al., 1994, 1995). In wild-type and ₆ +/ cerebella, the (318-400) antiserum precipitated the same number of muscimol binding sites (Fig. 6A). By this assay, the  protein was also present in both pure wild-type 129/Sv and pure C57BL/6 cerebella (data not shown). In contrast, immunoprecipitation of -containing receptors from ₆ / cerebella was greatly reduced (Fig. 6A). These data were extended by Western blot analysis of membrane protein samples isolated from individual +/+ and ₆ / cerebella. With use of a -subunit-specific antibody, (1-44)R7, raised against the N terminus (R. Pelz and W. Sieghart, unpublished data), ₆ / samples showed a dramatic reduction in the 53 ± 1 kDa  subunit band intensity to 25 ± 8% of +/+ levels (Fig. 6B). The residual  protein in the 6 / tissue had the same molecular weight as that in wild-type tissue (Fig. 6B).

---

Fig. 6. Immunoprecipitation and immunoblot analysis of GABA_A receptor  subunit levels in wild-type and ₆ / cerebella. A, After cerebellar GABA_A receptors were solubilized in Triton X-100/deoxycholate, the number of [³H]muscimol binding sites immunoprecipitated by the (318-400) antiserum from +/+, +/, and / cerebella was determined (n = 10-14). B, Immunoblot analysis: the marked  subunit reduction in ₆ / cerebella detected with the (1-44)R7 antiserum. The identity of the low molecular weight doublet (33 and 31 kDa) seen in all samples is unknown. C, A 51 kDa ₁ immunoreactive band is present in both / and +/+ cerebellar samples as detected with the 1(328-382)/1L antibody.
[View Larger Version of this Image (35K GIF file)]

---

Immunocytochemistry with the (1-44)R7 antibody clearly supported the Western blot and immunoprecipitation data (Fig. 7). Light microscopic immunocytochemistry with this antibody revealed a very intense cerebellar granule cell layer staining in wild-type animals (Fig. 7A), similar to that reported earlier using a different -specific antibody (Benke et al., 1991; Gao and Fritschy, 1995). The immunoreactivity originated mainly from staining of the glomeruli, and granule cell bodies were only weakly outlined (Fig. 7C). The glomeruli appeared as dark rings of labeled granule cell dendrites surrounding pale centers representing the unstained mossy fiber terminals (Fig. 7C). In contrast to the wild-type animals, in ₆ / mice the granule cell layer immunostaining for the  subunit was virtually absent (Fig. 7B). In particular, no immunoreactivity could be detected in the glomeruli (Fig. 7D), suggesting that ₆ / granule cell dendrites contain either no  subunit protein or an undetectably low level. Electron microscopic examination of the immunoreactivity for the  subunit in the granule cell layer further confirmed the lack of  subunit immunoreaction in ₆ / granule cells (not shown). Therefore, the residual  subunit immunoreactivity seen on Western blots may represent a level of protein undetectable by immunocytochemistry under our conditions, or it could come from cell types other than granule cells, because whole cerebella were used to prepare the protein extracts.

---

Fig. 7. Immunodetection of the  subunit of the GABA_A receptor in ₆ +/+ (A, C) or ₆ / (B, D) cerebella, using a polyclonal antibody  R7 and immunoperoxidase reaction. The granule cell layer showed intense immunoreactivity in ₆ +/+ animals but almost no staining was observed in the ₆ / mouse. C, At higher magnification, it is evident that the  subunit is localized mainly in the glomeruli, granule cell bodies (gc) being only weakly outlined. The glomeruli appear as dark rings of granule cell dendrites surrounding a pale center (arrows) representing the unstained mossy fiber terminal. D, In the ₆ / mice, both the granule cell bodies (gc) and the glomeruli (asterisks) are immunonegative for the  subunit. C and D were photographed using DIC optics. Scale bars: A, B, 500 µm; C, D, 10 µm.
[View Larger Version of this Image (107K GIF file)]

---

The  subunit loss occurs post-translationally

One possibility to explain the loss of  protein from ₆ / granule cells is through a change in regulation at the mRNA level; however, the  subunit mRNA level in the cerebellar granule cells was at normal levels when examined by in situ hybridization (Fig. 4E). High levels of  mRNA were seen in both wild-type and / granule cells.  mRNA expression was also examined in both pure 129/Sv and C57BL/6 strain wild-type animals and found not to differ (not shown). This result suggests that the loss of  subunit from the ₆ / cerebellar granule cells occurs post-translationally.

[³H]Muscimol and [³H]SR95531, two ligands that mark out ₆-and -containing GABA_A receptors

[³H]Muscimol and [³H]SR95531 are ligands that highlight restricted GABA molecule conformations (Sieghart, 1995). In particular, [³H]muscimol is the classic GABA_A ligand and has been used extensively for mapping GABA_A receptors in the brain (Palacios et al., 1980; Olsen et al., 1990). We used [³H]muscimol and [³H]SR95531 to autoradiographically probe the remaining GABA_A receptors in the  subunit-deficient/₆ / cerebellar granule cell layer. A clear cut, but completely unanticipated, pharmacological feature was revealed: the selective and extensive loss of high-affinity [³H]muscimol (Fig. 4B) and [³H]SR95531 (Fig. 4C) binding from the granule cells. Binding over the cerebellar molecular layer with these ligands remained unchanged, as did the levels of binding in the forebrain, e.g., normal levels of [³H]muscimol binding remain over the thalamus of / animals (Fig. 4B). The decrease in [³H]muscimol binding seen by autoradiography in ₆ / individuals was further quantified by studying [³H]muscimol binding to membranes from whole cerebella (Table 1). The level of high-affinity [³H]muscimol sites was reduced to ~25% of that found in control animals (Table 1). No significant reduction in [³H]muscimol binding was seen in +/ animals (data not shown). Saturation analysis revealed no change in the observed K_d values for [³H]muscimol in / animals (K_d is ~6 nM; Table 1). The residual binding is likely to come from sites within the molecular layer, the granule cell layer, and the deep cerebellar nuclei, all of which contain an _12/32 component. Under autoradiographic conditions, however, [³H]muscimol does not highlight these 1-containing receptors in the cerebellum. Rather, [³H]muscimol and [³H]SR95531 seem to selectively highlight ₆-containing receptors.

Evaluation of ₁ subunit levels in ₆ / and -deficient cerebella

The ₁ protein is expected to account for the majority of the remaining  subunits in the cerebellum of ₆ / mice (Sieghart, 1995; McKernan and Whiting, 1996; Wisden et al., 1996). To examine whether there was any concomitant change in the ₁-receptor population in the ₆ / cerebella, the portion of ₁ subunits complexed with the _2/3 and ₂ subunits was measured using three different ligands targeting the BZ binding site. These assays are not likely to measure any ₁ subunits that are complexed with the  but not the ₂ subunits, e.g., _12/3, because the GABA responses of these complexes cannot be modulated by BZs (Saxena and Macdonald, 1994).

Full saturation analysis was carried out on cerebellar membranes to determine the K_d and B_max values for [³H]Ro 15-1788 binding, diazepam-sensitive [³H]Ro 15-4513 binding, and [³H]zolpidem binding (Table 1). All three ligands identified approximately the same number of binding sites in the cerebellar membranes (~990-1160 fmol/mg protein). There was no statistically significant difference between the number of binding sites for any ligand between the ₆ +/+ and -deficient/₆ null groups, although the trend was always toward a reduced number of sites in the / animals. Total [³H]Ro 15-4513 binding sites (both diazepam-sensitive and -insensitive components, with the nonspecific binding being defined in the presence of 10 µM Ro 15-4513), however, were decreased by 44% in the / cerebella (Table 1), with a minor change in the affinity K_d constant. This figure is in line with the 30-40% contribution that the _62/32 component has been reported to make to the total Ro 15-4513 binding sites in the cerebellum (Sieghart et al., 1987; Turner et al., 1991; Korpi et al., 1993; Quirk et al., 1994).

These binding results suggest that the amount of total ₁ protein complexed with _2/3 and ₂ in the cerebellum is essentially unchanged between -deficient/₆ null and wild-type animals. Furthermore, immunocytochemistry with a polyclonal ₁-specific antibody (P16) showed no overt change in granule cell layer immunostaining at the light microscopic level in ₆ / cerebella compared with wild-type tissue (data not shown); however, this method may not pick out small changes in subunit levels. In fact, immunoblotting with ₁-specific antibodies did show a downward trend in ₁ protein levels between -deficient/₆ null and +/+ cerebellar samples (Fig. 6C). In / animals, a small reduction with high variability was seen in the ₁ 51 kDa band intensity, as determined by densitometric measurements. This was observed independently with three different ₁-specific antibodies: ₁(1-14) (S. Pollard and F. A. Stephenson, unpublished data), ₁(1-9) (Zezula and Sieghart, 1991), and ₁(328-382) (R. Pelz and W. Sieghart, unpublished data) (see Materials and Methods).

Behavioral characterization of ₆ null/-deficient mice

The total loss of ₆ from the cerebellum and the associated severe reduction of  subunit levels might be expected to have consequences for nervous system function in the mutant mice. The cerebellum integrates sensory input needed for maintaining balance and orientation, has a prominent role in the refinement of motor action, and may also participate in motor memory storage (Raymond et al., 1996). We looked for evidence of cerebellar-associated motor deficits in the / mice. In terms of simple observable behavior, mutant mice seem indistinguishable from wild-type littermates. The adult ₆ null/-deficient mice are active and agile, whether they be in the cage or roaming freely, and exhibit spontaneous activity, such as walking upside down on the ceiling of their cages. In an open field test, mutant mice showed as much exploratory activity as individuals with normal levels of ₆ and  proteins (Table 2). Mutant as well as wild-type mice learned the horizontal wire task (data not shown; see Materials and Methods). Both the wild-type and ₆ null/-deficient mice reached the rotating rod test learning criterion (Table 2). With these tests, we found no evidence to suggest any form of ataxia associated with cerebellar dysfunction. Additionally, a detailed behavioral analysis on an independently generated ₆ / mouse line, where the same exon was disrupted by insertion of a neo gene (exon 8, NcoI site) in a 129xC57BL/6 background, showed no abnormalities in motor behaviors (Gregg E. Homanics, Department of Anesthesiology, University of Pittsburgh, personal communication).

Table 2. Open field activity and rotating rod learning of wild-type and 6 / mice n Open field activity/5 min Number of crossings Number of fecal boli Number of rearings +/+ 10 96  ± 5 3.3  ± 0.7 6.7  ± 1.5  / 10 84  ± 18 3.2  ± 0.5 5.2  ± 1.1 n Rotarod performance: seconds on the rod per 180 sec/(percent of animals staying for 180 sec) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 +/+ 15 114  ± 11 (20%) 147  ± 9 (40%) 160  ± 11 (67%) 179  ± 1 (87%) 179  ± 1 (87%) 180  ± 0 (100%)  / 14 144  ± 11 (43%) 148  ± 12 (57%) 167  ± 8 (79%) 176  ± 3 (86%) 176  ± 4 (93%) 180  ± 0 (100%) Results are given as means ± SEM. No statistically significant differences (p < 0.05; Student's t test and repeated measures ANOVA). The Rotarod learning test was repeated with another batch of mice with similar results.

---

DISCUSSION

A mouse line lacking functional GABA_A receptor ₆ subunit protein has been generated. Because of the restricted ₆ gene expression profile, this mutation was expected to principally affect the cerebellum. Furthermore, in the cerebellum, the granule cell subunit protein level was markedly reduced relative to wild-type levels. Thus these mice effectively harbor a region-specific double subunit knock-out, and the GABA_A receptor complexity on granule cells is reduced to receptors largely containing just ₁, _2/3, and ₂ subunits. Two issues are discussed: the significance of multiple  subunits and defined assembly pathways for receptor subunits.

GABA_A receptor  subunit heterogeneity

Surprisingly, in spite of a large loss of granule cell GABA_A receptors, the ₆ null/-deficient mice are not grossly impaired in motor skills. This lack of phenotype under laboratory conditions was not anticipated from ₆ gene comparative studies. Both the conservation of peptide sequence in the N-terminal domain and a granule cell-specific expression pattern in the cerebellum of fish, birds, rodents, and humans imply that there has been a continual selection for the ₆ protein (Bahn et al., 1996; Hadingham et al., 1996).

In the ₆ null/-deficient mice, physiological changes in granule cell GABA_A receptors are expected, but these have not obviously impaired cerebellar function. Removal of two of the six subunits from granule cells will still leave functional receptors with _12/32 subunit combinations. Nevertheless, substitution of different  subunits in an _xx2 complex may influence the inhibitory postsynaptic current kinetics (Gingrich et al., 1995; Tia et al., 1996). Synaptic transmission at GABAergic synapses is generated by millisecond pulses of 0.5-1 mM GABA (reviewed by Mody et al., 1994). Under these conditions, recombinant _12/32 and _62/32 receptors do behave differently, with the ₆-containing receptors having a slower deactivation rate (Tia et al., 1996). The physiological role of the  subunit remains obscure (Shivers et al., 1989). During long applications of micromolar GABA, -subunits slow the acute macroscopic desensitization rate of recombinant GABA_A receptors (Saxena and Macdonald, 1994); however, this property has not been studied using fast, brief GABA application.

Selective subunit partnerships

The ₆ / mouse has provided insight into GABA_A receptor subunit assembly pathways in neurons. The ₆ protein derived from the targeted gene should terminate just after TM2. An analogous example has been studied for the mouse muscle nicotinic receptor  subunit. When truncated just after TM2 (M2) and co-expressed with wild-type nicotinic , , , and  subunits in COS cells, M2 interferes with receptor assembly (Verrall and Hall, 1992). Similarly, the truncated GABA_{A 6} protein (₆M2) may prevent ₆-containing receptors from reaching the granule cell surface. The association of ₆M2 and  may inhibit mature receptor expression by forming specific complexes in the endoplasmic reticulum that are not permissive for further receptor assembly and/or trafficking. These will be retained and degraded (Verrall and Hall, 1992; Connolly et al., 1996). As for the nicotinic acetylcholine and glycine receptors (Verrall and Hall, 1992; Kuhse et al., 1993; Sumikawa and Nishizaki, 1994; Kreienkamp et al., 1995), the information needed for specific assembly of the GABA_A receptor ₆ and  proteins is likely to be in their N-terminal domains, because the N-terminal domain of ₆ is sufficient to block  expression. Because they interact as an assembly intermediate, ₆ and  probably occur adjacent to each other in the mature receptor subunit ring.

There are several other scenarios. The ₆M2 polypeptide could be degraded before pairing with the  subunit. Because the subunit is not efficiently incorporated with other subunits, this might in turn be degraded. Alternatively, if no ₆ protein is present, the  mRNA might be translated inefficiently, implying that ₆ protein levels feed back to regulate the translation of  mRNA. Although this is an interesting possibility, there is no known mechanism.

Our results seem to confirm the antibody-based data suggesting that in vivo,  predominantly assembles with ₆ and not ₁ (Caruncho and Costa, 1994; Quirk et al., 1994; Caruncho et al., 1995). Nevertheless, from the genetic results alone, an ₆ and  interaction may be simply the first step allowing other subunits such as ₁ to subsequently join the complex. Thus, both ₁₆ or even ₁₆₂ might exist in vivo (Mertens et al., 1993; Pollard et al., 1995; R. Pelz and W. Sieghart, unpublished observations); however, because ₁, _2/3, and ₂ subunits cannot rescue the subunit in an ₆ / background, we predict that the _1x and _1x2 combinations will not be found to any great extent in vivo.

In recombinant systems [Xenopus oocytes, human embryonic kidney (HEK) 293 cells, and mouse L929 fibroblast cells], the situation is different. The  subunit can assemble to form functional receptors with either ₁ or ₆ as _1x, _6x, and possibly _1x2 complexes, with the exact  subunit used having little influence (Saxena and Macdonald, 1994, 1996; Ducic et al., 1995; Krishek et al., 1996). Thus there may be unique architectural editing or chaperone mechanisms present in granule neurons that are not found in Xenopus oocytes or HEK cells. Alternatively, the subunits may differ slightly in affinity for each other. In a recombinant system, the large amounts of protein present may allow many combinations to assemble, even if they have nonoptimal association parameters. The results presented here demonstrate the importance of studying subunit assembly pathways in the brain.

[³H]Muscimol as a selective autoradiographic probe for ₄, ₆, and  subunit associations

It has been suggested that under autoradiographic binding conditions the GABA_A site ligands [³H]muscimol and [³H]SR 95531 highlight a subpopulation of receptors in native membranes (Olsen et al., 1990). In a wide range of vertebrates, a hallmark of GABA_A sites in the CNS is the high levels of [³H]muscimol binding over the cerebellar granule cell layer (Palacios et al., 1980; Schmitz et al., 1988; Olsen et al., 1990; for review, see Wisden et al., 1996). A striking feature of our study was the almost total loss of high-affinity [³H]muscimol and [³H]SR95531 binding from the granule cell layer of ₆ null/-deficient cerebella (Fig. 4B,C), suggesting that the ₆ and/or  subunits are responsible for these ligand profiles. From recombinant data, the _6x subunit combination is insensitive to BZs (Saxena and Macdonald, 1996) and sensitive to GABA (EC₅₀ in the low micromolar range) but gives small currents (Saxena and Macdonald, 1994, 1996; Ducic et al., 1995). These properties would be consistent with the pharmacology of the cerebellar -containing receptors immunoprecipitated with a -specific antibody: high muscimol affinity and no BZ binding (Quirk et al., 1994).

Despite the absence of autoradiographic signal in / cerebella, muscimol is still an effective agonist of GABA_A receptors on cultured ₆ / granule cells (J. Mellor and A. D. Randall, unpublished observations), although electrophysiological assays most likely use the low-affinity site. The remaining _12/32 receptors in ₆ / granule cells should have a K_d for [³H]muscimol of ~5 nM (Lüddens et al., 1990) and could be expected to bind [³H]muscimol, but this is not the case under autoradiographic assay conditions. Similarly, in the inferior colliculi of wild-type and / animals, given the high concentration of ₁₂₂ receptors present (Persohn et al., 1992; Wisden et al., 1992; Fritschy and Möhler, 1995), it is difficult to explain the virtual absence of [³H]muscimol binding sites in autoradiography (Fig. 4B). There may be some factor specifically associated with _12/32 receptors on native membranes that prevents high-affinity [³H]muscimol binding.

As pointed out previously (Shivers et al., 1989; Laurie et al., 1992), both  subunit mRNA and protein closely parallel the distribution and abundance of [³H] muscimol binding (Fig. 4B,E), e.g., highest in cerebellar granule cells, followed by the thalamus, and then caudate-putamen, neocortex, and dentate gyrus (Shivers et al., 1989; Olsen et al., 1990; Benke et al., 1991). In the forebrain, ₄ subunit mRNA also largely follows  subunit distribution (compare Fig. 4, B and D; and see Wisden et al., 1992). The ₄ and ₆ subunits are closely related and form a subgroup set apart from the other  subunits (Seeburg et al., 1990; Ortells and Lunt, 1995; Tyndale et al., 1995). The total evidence suggests strongly that ₄ forms part of a GABA_A receptor that is an ₆ combination homolog. A phylogenetic clock comparison calculated that ₄ and ₆ are the oldest  subunits and that the  subunit is the oldest of all GABA_A receptor subunit genes (Ortells and Lunt, 1995). Therefore, a selective interaction of ₄ and ₆ with  might represent an early vertebrate GABA_A receptor subtype.

In conclusion, we have provided evidence for a specific association between the ₆ and  subunits in granule cell GABA_A receptors. It seems likely that similar assembly rules exist for other brain heteromeric ligand-gated channels, e.g., the neuronal nicotinic acetylcholine receptor (Vernallis et al., 1993) and ionotropic glutamate receptors.

---

FOOTNOTES

Received Oct. 10, 1996; revised Nov. 25, 1996; accepted Nov. 26, 1996.

^a   These authors made equally important contributions to different aspects of this work.
  

S.B. holds a European Community Human Capital and Mobility Fellowship (category 20). A.J.H.S. was supported by the Association for International Cancer Research. This work was supported in part by EC Grant BIO4-CT96-0585 to P.S. and W.S. We thank Terry Rabbitts, Theresa Langford, and Gareth King (Laboratory of Molecular Biology, Cambridge) for providing invaluable support and guidance with transgenic mice; Grayson Richards (F. Hoffman-LaRoche, Basel) for supplying Ro 15-1788; and Pirkko Johansson, Maija Sarviharju, and Antti Turhala (Department of Alcohol Research, National Public Health Institute, Helsinki) and Frances Emms (Merck Sharp and Dohme, Harlow) for technical help with behavior, autoradiography, and ligand binding. We are grateful to Gregg E. Homanics (Department of Anesthesiology, University of Pittsburgh) for communicating data on his ₆ knock-out mouse line before publication. Hilmar Bading, Louise Tierney, Stephen Hunt, Trevor Smart, and Nigel Unwin provided helpful comments on this manuscript.

Correspondence should be addressed to W. Wisden, Medical Research Council Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, UK.

---

REFERENCES

• Bahn S, Harvey RJ, Darlison MG, Wisden W (1996) Conservation of  aminobutyric acid type A receptor ₆ subunit gene expression in cerebellar granule cells. J Neurochem 66:1810-1818 . [Web of Science][Medline]
• Benke D, Mertens S, Trzecia A, Gillessen D, Möhler H (1991) Identification and immunohistochemical mapping of GABA_A receptor subtypes containing the  subunit in rat brain. FEBS Lett 283:145-149 . [Web of Science][Medline]
• Bonnerot C, Nicolas J-F (1993) Application of LacZ gene fusions to postimplantation development. Methods Enzymol 225:451-469 . [Web of Science][Medline]
• Caruncho HJ, Costa E (1994) Double-immunolabelling study of GABA_A receptor subunits in label-fracture replicas of cultured rat cerebellar granule cells. Receptors Channels 2:143-153 . [Web of Science][Medline]
• Caruncho HJ, Puia G, Möhler H, Costa E (1995) The density and distribution of six GABA_A receptor subunits in primary cultures of rat cerebellar granule cells. Neuroscience 67:583-593 . [Web of Science][Medline]
• Connolly CN, Krishek BJ, McDonald BJ, Smart TG, Moss SJ (1996) Assembly and cell surface expression of heteromeric and homomeric -aminobutyric acid type A receptors. J Biol Chem 271:89-96 . [Abstract/Free Full Text]
• Ducic I, Caruncho HJ, Zhu WJ, Vicini S, Costa E (1995) -aminobutyric acid gating of Cl channels in recombinant GABA_A receptors. J Pharmacol Exp Ther 272:438-445 . [Abstract/Free Full Text]
• Fritschy J-M, Möhler H (1995) GABA_A-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359:154-194 . [Web of Science][Medline]
• Gao B, Fritschy J-M (1995) Cerebellar granule cells in vitro recapitulate the in vivo pattern of GABA_A-receptor subunit expression. Dev Brain Res 88:1-16 . [Medline]
• Gingrich KJ, Roberts WA, Kass RS (1995) Dependence of the GABA_A receptor gating kinetics on the -subunit isoform: implications for structure-function relations and synaptic transmission. J Physiol (Lond) 489:529-543 . [Abstract/Free Full Text]
• Green WN, Claudio T (1993) Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially. Cell 74:57-69 . [Web of Science][Medline]
• Günther U, Benson J, Benke D, Fritschy J-M, Reyes G, Knoflach F, Crestini F, Aguzzi A, Arigoni M, Lang Y, Bluethmann H, Möhler H, Lüscher B (1995) Benzodiazepine-insensitive mice generated by targeted disruption of the ₂ subunit gene of -aminobutyric acid type A receptors. Proc Natl Acad Sci USA 92:7749-7753 . [Abstract/Free Full Text]
• Gutiérrez A, Khan ZU, De Blas AL (1996) Immunocytochemical localization of the ₆ subunit of the -aminobutyric acid_A receptor in the rat nervous system. J Comp Neurol 365:504-510 . [Web of Science][Medline]
• Hadingham KL, Garrett EM, Wafford KA, Bain C, Heavens RP, Sirinathsinghji DJS, Whiting PJ (1996) Cloning of cDNAs encoding the human -aminobutyric acid type A receptor 6 subunit and characterization of the pharmacology of ₆-containing receptors. Mol Pharmacol 49:253-259 . [Abstract]
• Jones A, Bahn S, Grant AL, Köhler M, Wisden W (1996) Characterization of a cerebellar granule cell-specific gene encoding the -aminobutyric acid type A receptor ₆ subunit. J Neurochem 67:907-916 . [Web of Science][Medline]
• Khan ZU, Gutiérrez A, De Blas AL (1994) The subunit composition of a GABA_A/benzodiazepine receptor from rat cerebellum. J Neurochem 63:371-374 . [Web of Science][Medline]
• Khan ZU, Gutiérrez A, De Blas AL (1996) The ₁ and ₆ subunits can coexist in the same cerebellar GABA_A receptor maintaining their individual benzodiazepine-binding specificities. J Neurochem 66:685-691 . [Web of Science][Medline]
• Korpi ER, Lüddens H (1993) Regional -aminobutyric acid sensitivity of t-butylbicyclophosphoro[³⁵S]thionate binding depends on -aminobutyric acid_A receptor  subunit. Mol Pharmacol 44:87-92 . [Abstract]
• Korpi ER, Uusi-Oukari M, Kaivola J (1993) Postnatal development of diazepam-insensitive [³H]Ro 15-4513 binding sites. Neuroscience 53:483-488 . [Web of Science][Medline]
• Korpi ER, Kuner T, Seeburg PH, Lüddens H (1995) Selective antagonist for the cerebellar granule cell-specific -aminobutyric acid type A receptor. Mol Pharmacol 47:283-289 . [Abstract]
• Kreienkamp H-J, Maeda RK, Sine SM, Taylor P (1995) Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor. Neuron 14:635-644 . [Web of Science][Medline]
• Krishek BJ, Amato A, Connolly CN, Moss SJ, Smart TG (1996) Proton sensitivity of the GABA_A receptor is associated with the receptor subunit composition. J Physiol (Lond) 492:431-443 . [Abstract/Free Full Text]
• Kuhse J, Laube B, Magalei D, Betz H (1993) Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron 11:1049-1056 . [Web of Science][Medline]
• Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of thirteen GABA_A receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci 12:1063-1076 . [Abstract]
• Lüddens H, Pritchett DB, Köhler M, Killisch I, Keinänen K, Monyer H, Sprengel R, Seeburg PH (1990) Cerebellar GABA_A receptor selective for behavioural alcohol antagonist. Nature 346:648-651 . [Medline]
• Malminiemi O, Korpi ER (1989) Diazepam-insensitive [³H]Ro 15-4513 binding in intact cultured cerebellar granule cells. Eur J Pharmacol 169:53-60 . [Web of Science][Medline]
• Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348-352 . [Medline]
• McKernan RM, Whiting PJ (1996) Which GABA_A-receptor subtypes really occur in the brain? Trends Neurosci 19:139-143 . [Web of Science][Medline]
• Mertens S, Benke D, Möhler H (1993) GABA_A receptor populations with novel subunit combinations and drug-binding profiles identified in brain by ₅- and -subunit-specific immunoprecipitation. J Biol Chem 268:5965-5973 . [Abstract/Free Full Text]
• Mody I, De Koninck Y, Otis TS, Soltez I (1994) Bridging the cleft at GABA synapses in the brain. Trends Neurosci 17:517-525 . [Web of Science][Medline]
• Mossier B, Tögel M, Fuchs K, Sieghart W (1994) Immunoaffinity purification of -aminobutyric acidA (GABA_A) receptors containing ₁-subunits: evidence for the presence of a single type of -subunit in GABA_A receptors. J Biol Chem 269:25777-25782 . [Abstract/Free Full Text]
• Mountford P, Zevnik B, Düwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A (1994) Discistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA 91:4303-4307 . [Abstract/Free Full Text]
• Nehls M, Kyewski B, Messerle M, Waldschütz R, Schüddekopf K, Smith AJH, Boehm T (1996) Two genetically separable steps in the differentiation of thymic epithelium. Science 272:886-889 . [Abstract]
• Nusser Z, Roberts JDB, Baude A, Richards JG, Somogyi P (1995) Relative densities of synaptic and extrasynaptic GABA_A receptors on cerebellar granule cells as determined by a quantitative immunogold method. J Neurosci 15:2948-2960 . [Abstract]
• Nusser Z, Sieghart W, Stephenson FA, Somogyi P (1996) The ₆ subunit of the GABA_A receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. J Neurosci 16:103-114 . [Abstract/Free Full Text]
• Olsen RW, McCabe RT, Wamsley JK (1990) GABA_A receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system. J Chem Neuroanat 3:59-76 . [Web of Science][Medline]
• Ortells MO, Lunt GG (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci 18:121-127 . [Web of Science][Medline]
• Palacios JM, Young WS, Kuhar MJ (1980) Autoradiographic localization of -aminobutyric acid (GABA) receptors in rat cerebellum. Proc Natl Acad Sci USA 77:670-679 . [Abstract/Free Full Text]
• Persohn E, Malherbe P, Richards JG (1992) Comparative molecular neuroanatomy of cloned GABA_A receptor subunits in the rat CNS. J Comp Neurol 326:193-216 . [Web of Science][Medline]
• Pollard S, Thompson CL, Stephenson FA (1995) Quantitative characterization of ₆ and ₁₆ subunit-containing native -aminobutyric acid_A receptors of adult rat cerebellum demonstrates two  subunits per receptor oligomer. J Biol Chem 270:21285-21290 . [Abstract/Free Full Text]
• Quirk K, Gillard NP, Ragan CI, Whiting PJ, McKernan RM (1994) Model of subunit composition of -aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their  and / subunits. J Biol Chem 269:16020-16028 . [Abstract/Free Full Text]
• Quirk K, Whiting PJ, Ragan CI, McKernan RM (1995) Characterization of -containing GABA_A receptors from the rat brain. Eur J Pharmacol 290:175-181 . [Web of Science][Medline]
• Randall AD, Tsien RW (1995) Pharmacological dissection of multiple classes of Ca²⁺ channel currents in rat cerebellar granule cells. J Neurosci 15:2995-3012. [Abstract]
• Raymond JL, Lisberger SG, Mauk MD (1996) The cerebellum: a neuronal learning machine? Science 272:1126-1130 . [Abstract]
• Santi MR, Vicini S, Eldadah B, Neale JH (1994) Analysis by polymerase chain reaction of ₁ and ₆ GABA_A receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings. J Neurochem 63:2357-2360 . [Web of Science][Medline]
• Saxena NC, Macdonald RL (1994) Assembly of GABA_A receptor subunits: role of the  subunit. J Neurosci 14:7077-7086 . [Abstract]
• Saxena NC, Macdonald RL (1996) Properties of putative cerebellar -aminobutyric acid_A receptor isoforms. Mol Pharmacol 49:567-579 . [Abstract]
• Schmitz E, Reichelt R, Walkowiak W, Richards JG, Hebebrand J (1988) A comparative phylogenetic study of the distribution of cerebellar GABA_A/benzodiazepine receptors using radioligands and monoclonal antibodies. Brain Res 473:314-320 . [Web of Science][Medline]
• Seeburg PH, Wisden W, Verdoorn TA, Pritchett DB, Werner P, Herb A, Lüddens H, Sprengel R, Sakmann B (1990) The GABA_A receptor family: molecular and functional diversity. Cold Spring Harb Symp Quant Biol 55:29-40 . [Abstract/Free Full Text]
• Shivers BD, Killisch I, Sprengel R, Sontheimer H, Köhler M, Schofield PR, Seeburg PH (1989) Two novel GABA_A receptor subunits exist in distinct neuronal subpopulations. Neuron 3:327-337 . [Web of Science][Medline]
• Sieghart W (1995) Structure and pharmacology of -aminobutyric acid_A receptor subtypes. Pharmacol Rev 47:181-234 . [Web of Science][Medline]
• Sieghart W, Eichinger A, Richards JG, Möhler H (1987) Photoaffinity labelling of benzodiazepine receptor proteins with the partial inverse agonist [³H]Ro 15-4513: a biochemical and autoradiographic study. J Neurochem 48:46-52 . [Web of Science][Medline]
• Smith AJH, De Sousa MA, Kwabi-Addo B, Heppell-Parton A, Impey H, Rabbitts P (1995) A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature Genet 9:376-385. [Web of Science][Medline]
• Somogyi P, Takagi H, Richards JG, Möhler H (1989) Subcellular localization of benzodiazepine/GABA_A receptors in the cerebellum of rat, cat, and monkey using monoclonal antibodies. J Neurosci 9:2197-2209 . [Abstract]
• Somogyi P, Fritschy J-M, Benke D, Roberts JDB, Sieghart W (1996) The ₂ subunit of the GABA_A receptor is concentrated in synaptic junctions containing the ₁ and _2/3 subunits in hippocampus, cerebellum and globus pallidus. Neuropharmacology, in press.
• Stephenson FA (1995) The GABA_A receptors. Biochem J 310:1-9 .
• Sumikawa K, Nishizaki T (1994) The amino acid residues 1-128 in the  subunit of the nicotinic acetylcholine receptor contain assembly signals. Mol Brain Res 25:257-264 . [Medline]
• Thompson CL, Bodewitz G, Stephenson FA, Turner JD (1992) Mapping of GABA_A receptor ₅ and ₆ subunit-like immunoreactivity in rat brain. Neurosci Lett 144:53-56 . [Web of Science][Medline]
• Tia S, Wang JF, Kotchabhakdi N, Vicini S (1996) Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABA_A receptor ₆ subunit. J Neurosci 16:3630-3640 . [Abstract/Free Full Text]
• Tögel M, Mossier B, Fuchs K, Sieghart W (1994) -Aminobutyric acid_A receptors displaying association of ₃-subunits with _2/3 and different -subunits exhibit unique pharmacological properties. J Biol Chem 269:12993-12998 . [Abstract/Free Full Text]
• Turner DM, Sapp DW, Olsen RW (1991) The benzodiazepine/alcohol antagonist Ro 15-4513: binding to a GABA_A receptor subtype that is insensitive to diazepam. J Pharmacol Exp Ther 257:1236-1242 . [Abstract/Free Full Text]
• Tyndale RF, Olsen RW, Tobin AJ (1995) GABA_A receptors. In: Handbook of receptors and channels: ligand- and voltage-gated ion channels (North RA, ed), pp 265-290. Boca Raton, FL: CRC.
• Varecka L, Wu C-H, Rotter A, Frostholm A (1994) GABA_A/benzodiazepine receptor ₆ subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice. J Comp Neurol 339:341-352 . [Web of Science][Medline]
• Vernallis AB, Conroy WG, Berg DG (1993) Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron 10:451-464 . [Web of Science][Medline]
• Verrall S, Hall ZW (1992) The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell 68:23-31 . [Web of Science][Medline]
• Wisden W, Morris BJ (1994) In situ hybridization with synthetic oligonucleotide probes. In: In situ hybridization protocols for the brain (Wisden W, Morris BJ, eds), pp 9-34. London: Academic.
• Wisden W, Herb A, Wieland H, Keinänen K, Lüddens H, Seeburg PH (1991) Cloning, pharmacological characteristics and expression pattern of the rat GABA_A receptor ₄ subunit. FEBS Lett 289:227-230 . [Web of Science][Medline]
• Wisden W, Laurie DJ, Monyer H, Seeburg PH (1992) The distribution of thirteen GABA_A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12:1040-1062 . [Abstract]
• Wisden W, Korpi ER, Bahn S (1996) The cerebellum: a model system for studying GABA_A receptor diversity. Neuropharmacology 35:1139-1160. [Web of Science][Medline]
• Wong G, Sarviharju M, Toropainen M, Matecka D, Korpi ER (1996) Pharmacological actions of subtype-selective and novel GABAergic ligands in rat lines with differential sensitivity to ethanol. Pharmacol Biochem Behav 53:723-730 . [Web of Science][Medline]
• Zezula J, Sieghart W (1991) Isolation of type I and type II GABA_A-benzodiazepine receptors by immunoaffinity chromatography. FEBS Lett 284:15-18 . [Web of Science][Medline]

---

Copyright ©1997 Society for Neuroscience   0270-6474/1997/171350-13$05.00/0

This article has been cited by other articles:

				K. H. Kaur, R. Baur, and E. Sigel Unanticipated Structural and Functional Properties of {delta}-Subunit-containing GABAA Receptors J. Biol. Chem., March 20, 2009; 284(12): 7889 - 7896. [Abstract] [Full Text] [PDF]

				S. L. Planey, S. K. Keay, C.-O. Zhang, and D. A. Zacharias Palmitoylation of Cytoskeleton Associated Protein 4 by DHHC2 Regulates Antiproliferative Factor-mediated Signaling Mol. Biol. Cell, March 1, 2009; 20(5): 1454 - 1463. [Abstract] [Full Text] [PDF]

				E. Sanna, M. C. Mostallino, L. Murru, M. Carta, G. Talani, S. Zucca, M. L. Mura, E. Maciocco, and G. Biggio Changes in Expression and Function of Extrasynaptic GABAA Receptors in the Rat Hippocampus during Pregnancy and after Delivery J. Neurosci., February 11, 2009; 29(6): 1755 - 1765. [Abstract] [Full Text] [PDF]

				H. Matsuoka, K. Harada, Y. Endo, A. Warashina, Y. Doi, J. Nakamura, and M. Inoue Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells J. Physiol., October 15, 2008; 586(20): 4825 - 4842. [Abstract] [Full Text] [PDF]

				A.-M. Linden, M. I. Aller, E. Leppa, P. H. Rosenberg, W. Wisden, and E. R. Korpi K+ Channel TASK-1 Knockout Mice Show Enhanced Sensitivities to Ataxic and Hypnotic Effects of GABAA Receptor Ligands J. Pharmacol. Exp. Ther., October 1, 2008; 327(1): 277 - 286. [Abstract] [Full Text] [PDF]

				A. Lorincz and Z. Nusser Specificity of Immunoreactions: The Importance of Testing Specificity in Each Method J. Neurosci., September 10, 2008; 28(37): 9083 - 9086. [Full Text] [PDF]

				R. W. Olsen and W. Sieghart International Union of Pharmacology. LXX. Subtypes of {gamma}-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update Pharmacol. Rev., September 1, 2008; 60(3): 243 - 260. [Abstract] [Full Text] [PDF]

				W. Hevers, S. H. Hadley, H. Luddens, and J. Amin Ketamine, But Not Phencyclidine, Selectively Modulates Cerebellar GABAA Receptors Containing {alpha}6 and {delta} Subunits J. Neurosci., May 14, 2008; 28(20): 5383 - 5393. [Abstract] [Full Text] [PDF]

				Y.-H. Hsieh, T. E. Dick, and R. E. Siegel Adaptation to hypobaric hypoxia involves GABAA receptors in the pons Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R549 - R557. [Abstract] [Full Text] [PDF]

				H. L. Payne, W. M. Connelly, J. H. Ives, R. Lehner, B. Furtmuller, W. Sieghart, P. Tiwari, J. M. Lucocq, G. Lees, and C. L. Thompson GABAA {alpha}6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer J. Biol. Chem., October 5, 2007; 282(40): 29130 - 29143. [Abstract] [Full Text] [PDF]

				R. P. Bonin, L. J. Martin, J. F. MacDonald, and B. A. Orser {alpha}5GABAA Receptors Regulate the Intrinsic Excitability of Mouse Hippocampal Pyramidal Neurons J Neurophysiol, October 1, 2007; 98(4): 2244 - 2254. [Abstract] [Full Text] [PDF]

				S. G. Brickley, M. I. Aller, C. Sandu, E. L. Veale, F. G. Alder, H. Sambi, A. Mathie, and W. Wisden TASK-3 Two-Pore Domain Potassium Channels Enable Sustained High-Frequency Firing in Cerebellar Granule Neurons J. Neurosci., August 29, 2007; 27(35): 9329 - 9340. [Abstract] [Full Text] [PDF]

				L. Quignodon, C. Grijota-Martinez, E. Compe, R. Guyot, N. Allioli, D. Laperriere, R. Walker, P. Meltzer, S. Mader, J. Samarut, et al. A combined approach identifies a limited number of new thyroid hormone target genes in post-natal mouse cerebellum J. Mol. Endocrinol., July 1, 2007; 39(1): 17 - 28. [Abstract] [Full Text] [PDF]

				S. H. Hadley and J. Amin Rat {alpha}6beta2{delta} GABAA receptors exhibit two distinct and separable agonist affinities J. Physiol., June 15, 2007; 581(3): 1001 - 1018. [Abstract] [Full Text] [PDF]

				R. L. Fradley, M. R. Guscott, S. Bull, D. J. Hallett, S. C. Goodacre, K. A. Wafford, E. M. Garrett, R. J. Newman, G. F. O'Meara, P. J. Whiting, et al. Differential contribution of GABAA receptor subtypes to the anticonvulsant efficacy of benzodiazepine site ligands J Psychopharmacol, June 1, 2007; 21(4): 384 - 391. [Abstract] [PDF]

				E. Charrier, B. Mosinger, C. Meissirel, M. Aguera, V. Rogemond, S. Reibel, P. Salin, N. Chounlamountri, V. Perrot, M.-F. Belin, et al. Transient alterations in granule cell proliferation, apoptosis and migration in postnatal developing cerebellum of CRMP1-/- mice Genes Cells, December 1, 2006; 11(12): 1337 - 1352. [Abstract] [Full Text] [PDF]

				D. Chandra, F. Jia, J. Liang, Z. Peng, A. Suryanarayanan, D. F. Werner, I. Spigelman, C. R. Houser, R. W. Olsen, N. L. Harrison, et al. GABAA receptor {alpha}4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol PNAS, October 10, 2006; 103(41): 15230 - 15235. [Abstract] [Full Text] [PDF]

				S. F. Maison, T. W. Rosahl, G. E. Homanics, and M. C. Liberman Functional Role of GABAergic Innervation of the Cochlea: Phenotypic Analysis of Mice Lacking GABAA Receptor Subunits {alpha}1, {alpha}2, {alpha}5, {alpha}6, beta2, beta3, or {delta} J. Neurosci., October 4, 2006; 26(40): 10315 - 10326. [Abstract] [Full Text] [PDF]

				P. I. Ortinski, J. R. Turner, A. Barberis, G. Motamedi, R. P. Yasuda, B. B. Wolfe, K. J. Kellar, and S. Vicini Deletion of the GABAA Receptor {alpha}1 Subunit Increases Tonic GABAA Receptor Current: A Role for GABA Uptake Transporters J. Neurosci., September 6, 2006; 26(36): 9323 - 9331. [Abstract] [Full Text] [PDF]

				S. Nakanishi and M. Okazawa Membrane potential-regulated Ca2+ signalling in development and maturation of mammalian cerebellar granule cells J. Physiol., September 1, 2006; 575(2): 389 - 395. [Abstract] [Full Text] [PDF]

				K. R. Drasbek and K. Jensen THIP, a Hypnotic and Antinociceptive Drug, Enhances an Extrasynaptic GABAA Receptor-mediated Conductance in Mouse Neocortex Cereb Cortex, August 1, 2006; 16(8): 1134 - 1141. [Abstract] [Full Text] [PDF]

				V. Santhakumar, H. J. Hanchar, M. Wallner, R. W. Olsen, and T. S. Otis Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene. J. Neurosci., March 22, 2006; 26(12): 3357 - 3364. [Abstract] [Full Text] [PDF]

				S. i Storustovu and B. Ebert Pharmacological Characterization of Agonists at {delta}-Containing GABAA Receptors: Functional Selectivity for Extrasynaptic Receptors Is Dependent on the Absence of {gamma}2 J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1351 - 1359. [Abstract] [Full Text] [PDF]

				M. I. Aller, E. L. Veale, A.-M. Linden, C. Sandu, M. Schwaninger, L. J. Evans, E. R. Korpi, A. Mathie, W. Wisden, and S. G. Brickley Modifying the Subunit Composition of TASK Channels Alters the Modulation of a Leak Conductance in Cerebellar Granule Neurons J. Neurosci., December 7, 2005; 25(49): 11455 - 11467. [Abstract] [Full Text] [PDF]

				F. Jia, L. Pignataro, C. M. Schofield, M. Yue, N. L. Harrison, and P. A. Goldstein An Extrasynaptic GABAA Receptor Mediates Tonic Inhibition in Thalamic VB Neurons J Neurophysiol, December 1, 2005; 94(6): 4491 - 4501. [Abstract] [Full Text] [PDF]

				I. Mody Aspects of the homeostaic plasticity of GABAA receptor-mediated inhibition J. Physiol., January 1, 2005; 562(1): 37 - 46. [Abstract] [Full Text] [PDF]

				J. K. Christensen, A. V. Paternain, S. Selak, P. K. Ahring, and J. Lerma A Mosaic of Functional Kainate Receptors in Hippocampal Interneurons J. Neurosci., October 13, 2004; 24(41): 8986 - 8993. [Abstract] [Full Text] [PDF]

				W. Wei, L. C. Faria, and I. Mody Low Ethanol Concentrations Selectively Augment the Tonic Inhibition Mediated by {delta} Subunit-Containing GABAA Receptors in Hippocampal Neurons J. Neurosci., September 22, 2004; 24(38): 8379 - 8382. [Abstract] [Full Text] [PDF]

				V. B. Caraiscos, E. M. Elliott, K. E. You-Ten, V. Y. Cheng, D. Belelli, J. G. Newell, M. F. Jackson, J. J. Lambert, T. W. Rosahl, K. A. Wafford, et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by {alpha}5 subunit-containing {gamma}-aminobutyric acid type A receptors PNAS, March 9, 2004; 101(10): 3662 - 3667. [Abstract] [Full Text] [PDF]

				M. Wallner, H. J. Hanchar, and R. W. Olsen From The Cover: Ethanol enhances {alpha}4{beta}3{delta} and {alpha}6{beta}3{delta} {gamma}-aminobutyric acid type A receptors at low concentrations known to affect humans PNAS, December 9, 2003; 100(25): 15218 - 15223. [Abstract] [Full Text] [PDF]

				B. M. Stell, S. G. Brickley, C. Y. Tang, M. Farrant, and I. Mody Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by {delta} subunit-containing GABAA receptors PNAS, November 25, 2003; 100(24): 14439 - 14444. [Abstract] [Full Text] [PDF]

				W. Wei, N. Zhang, Z. Peng, C. R. Houser, and I. Mody Perisynaptic Localization of {delta} Subunit-Containing GABAA Receptors and Their Activation by GABA Spillover in the Mouse Dentate Gyrus J. Neurosci., November 19, 2003; 23(33): 10650 - 10661. [Abstract] [Full Text] [PDF]

				L. Bouslama-Oueghlani, R. Wehrle, C. Sotelo, and I. Dusart The Developmental Loss of the Ability of Purkinje Cells to Regenerate Their Axons Occurs in the Absence of Myelin: An In Vitro Model to Prevent Myelination J. Neurosci., September 10, 2003; 23(23): 8318 - 8329. [Abstract] [Full Text] [PDF]

				G. B. Richerson and Y. Wu Dynamic Equilibrium of Neurotransmitter Transporters: Not Just for Reuptake Anymore J Neurophysiol, September 1, 2003; 90(3): 1363 - 1374. [Abstract] [Full Text] [PDF]

				I. Spigelman, Z. Li, J. Liang, E. Cagetti, S. Samzadeh, R. M. Mihalek, G. E. Homanics, and R. W. Olsen Reduced Inhibition and Sensitivity to Neurosteroids in Hippocampus of Mice Lacking the GABAA Receptor {delta} Subunit J Neurophysiol, August 1, 2003; 90(2): 903 - 910. [Abstract] [Full Text] [PDF]

				Y. A. Blednov, D. Walker, H. Alva, K. Creech, G. Findlay, and R. A. Harris GABAA Receptor {alpha}1 and {beta}2 Subunit Null Mutant Mice: Behavioral Responses to Ethanol J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 854 - 863. [Abstract] [Full Text] [PDF]

				A. C. Engblom, F. F. Johansen, and U. Kristiansen Actions and Interactions of Extracellular Potassium and Kainate on Expression of 13 gamma -Aminobutyric Acid Type A Receptor Subunits in Cultured Mouse Cerebellar Granule Neurons J. Biol. Chem., May 2, 2003; 278(19): 16543 - 16550. [Abstract] [Full Text] [PDF]

				Y. A. Blednov, S. Jung, H. Alva, D. Wallace, T. Rosahl, P.-J. Whiting, and R. A. Harris Deletion of the alpha 1 or beta 2 Subunit of GABAA Receptors Reduces Actions of Alcohol and Other Drugs J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 30 - 36. [Abstract] [Full Text] [PDF]

				T. A. Simeone, S. D. Donevan, and J. M. Rho Molecular Biology and Ontogeny of {gamma}-Aminobutyric Acid (GABA) Receptors in the Mammalian Central Nervous System J Child Neurol, January 1, 2003; 18(1): 39 - 48. [Abstract] [PDF]

				N. Collinson, F. M. Kuenzi, W. Jarolimek, K. A. Maubach, R. Cothliff, C. Sur, A. Smith, F. M. Otu, O. Howell, J. R. Atack, et al. Enhanced Learning and Memory and Altered GABAergic Synaptic Transmission in Mice Lacking the alpha 5 Subunit of the GABAA Receptor J. Neurosci., July 1, 2002; 22(13): 5572 - 5580. [Abstract] [Full Text] [PDF]

				J. L. Fisher Amiloride Inhibition of gamma -Aminobutyric AcidA Receptors Depends upon the alpha Subunit Subtype Mol. Pharmacol., June 1, 2002; 61(6): 1322 - 1328. [Abstract] [Full Text] [PDF]

				T. J. Jentsch, V. Stein, F. Weinreich, and A. A. Zdebik Molecular Structure and Physiological Function of Chloride Channels Physiol Rev, April 1, 2002; 82(2): 503 - 568. [Abstract] [Full Text] [PDF]

				Z. Nusser, L. M. Kay, G. Laurent, G. E. Homanics, and I. Mody Disruption of GABAA Receptors on GABAergic Interneurons Leads to Increased Oscillatory Power in the Olfactory Bulb Network J Neurophysiol, December 1, 2001; 86(6): 2823 - 2833. [Abstract] [Full Text] [PDF]

				C. Sur, K. A. Wafford, D. S. Reynolds, K. L. Hadingham, F. Bromidge, A. Macaulay, N. Collinson, G. O'Meara, O. Howell, R. Newman, et al. Loss of the Major GABAA Receptor Subtype in the Brain Is Not Lethal in Mice J. Neurosci., May 15, 2001; 21(10): 3409 - 3418. [Abstract] [Full Text] [PDF]

				B. J Krishek and T. G Smart Proton sensitivity of rat cerebellar granule cell GABAA receptors: dependence on neuronal development J. Physiol., January 15, 2001; 530(2): 219 - 233. [Abstract] [Full Text] [PDF]

				M. Kneussel, J. H. Brandstatter, B. Laube, S. Stahl, U. Muller, and H. Betz Loss of Postsynaptic GABAA Receptor Clustering in Gephyrin-Deficient Mice J. Neurosci., November 1, 1999; 19(21): 9289 - 9297. [Abstract] [Full Text] [PDF]

				R. M. Mihalek, P. K. Banerjee, E. R. Korpi, J. J. Quinlan, L. L. Firestone, Z.-P. Mi, C. Lagenaur, V. Tretter, W. Sieghart, S. G. Anagnostaras, et al. Attenuated sensitivity to neuroactive steroids in gamma -aminobutyrate type A receptor delta subunit knockout mice PNAS, October 26, 1999; 96(22): 12905 - 12910. [Abstract] [Full Text] [PDF]

				A. R. Brooks-Kayal, M. D. Shumate, H. Jin, D. D. Lin, T. Y. Rikhter, K. L. Holloway, and D. A. Coulter Human Neuronal gamma -Aminobutyric AcidA Receptors: Coordinated Subunit mRNA Expression and Functional Correlates in Individual Dentate Granule Cells J. Neurosci., October 1, 1999; 19(19): 8312 - 8318. [Abstract] [Full Text] [PDF]

				S. Gustincich, A. Feigenspan, W. Sieghart, and E. Raviola Composition of the GABAA Receptors of Retinal Dopaminergic Neurons J. Neurosci., September 15, 1999; 19(18): 7812 - 7822. [Abstract] [Full Text] [PDF]

				E. Bencsits, V. Ebert, V. Tretter, and W. Sieghart A Significant Part of Native gamma -Aminobutyric AcidA Receptors Containing alpha 4 Subunits Do Not Contain gamma or delta Subunits J. Biol. Chem., July 9, 1999; 274(28): 19613 - 19616. [Abstract] [Full Text] [PDF]

				C. Sur, S. J. Farrar, J. Kerby, P. J. Whiting, J. R. Atack, and R. M. McKernan Preferential Coassembly of alpha 4 and delta  Subunits of the gamma -Aminobutyric AcidA Receptor in Rat Thalamus Mol. Pharmacol., July 1, 1999; 56(1): 110 - 115. [Abstract] [Full Text]

				S. G. Brickley, S. G. Cull-Candy, and M. Farrant Single-Channel Properties of Synaptic and Extrasynaptic GABAA Receptors Suggest Differential Targeting of Receptor Subtypes J. Neurosci., April 15, 1999; 19(8): 2960 - 2973. [Abstract] [Full Text] [PDF]

				P. Poisbeau, M. C. Cheney, M. D. Browning, and I. Mody Modulation of Synaptic GABAA Receptor Function by PKA and PKC in Adult Hippocampal Neurons J. Neurosci., January 15, 1999; 19(2): 674 - 683. [Abstract] [Full Text] [PDF]

				K. F Haas and R. L Macdonald GABAA receptor subunit {gamma}2 and {delta} subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts J. Physiol., January 1, 1999; 514(1): 27 - 45. [Abstract] [Full Text] [PDF]

				T.-J. Zhao, M. Li, T. H. Chiu, and H. C. Rosenberg Decreased Benzodiazepine Binding with Little Effect on gamma -Aminobutyric Acid Binding in Rat Brain After Treatment with Antisense Oligodeoxynucleotide to the gamma -Aminobutyric AcidA Receptor Gamma-2 Subunit J. Pharmacol. Exp. Ther., November 1, 1998; 287(2): 752 - 759. [Abstract] [Full Text]

				M. R. PICCIOTTO and K. WICKMAN Using Knockout and Transgenic Mice to Study Neurophysiology and Behavior Physiol Rev, October 1, 1998; 78(4): 1131 - 1163. [Abstract] [Full Text] [PDF]

				J. A. van Hooft, A. D. Spier, J. L. Yakel, S. C. R. Lummis, and H. P. M. Vijverberg Promiscuous coassembly of serotonin 5-HT3 and nicotinic alpha 4 receptor subunits into Ca2+-permeable ion channels PNAS, September 15, 1998; 95(19): 11456 - 11461. [Abstract] [Full Text] [PDF]

				E. R. Korpi, R. Makela, and M. Uusi-Oukari Ethanol: Novel Actions on Nerve Cell Physiology Explain Impaired Functions Physiology, August 1, 1998; 13(4): 164 - 170. [Abstract] [Full Text] [PDF]

				E. A. Barnard, P. Skolnick, R. W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup, A. N. Bateson, and S. Z. Langer International Union of Pharmacology. XV. Subtypes of gamma -Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Structure and Receptor Function Pharmacol. Rev., June 1, 1998; 50(2): 291 - 314. [Abstract] [Full Text] [PDF]

				J. R. Mellor, D. Merlo, A. Jones, W. Wisden, and A. D. Randall Mouse Cerebellar Granule Cell Differentiation: Electrical Activity Regulates the GABAA Receptor alpha 6 Subunit Gene J. Neurosci., April 15, 1998; 18(8): 2822 - 2833. [Abstract] [Full Text] [PDF]

				M. Jechlinger, R. Pelz, V. Tretter, T. Klausberger, and W. Sieghart Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABAA Receptors Containing alpha 6 Subunits J. Neurosci., April 1, 1998; 18(7): 2449 - 2457. [Abstract] [Full Text] [PDF]

				Z. Nusser, W. Sieghart, and P. Somogyi Segregation of Different GABAA Receptors to Synaptic and Extrasynaptic Membranes of Cerebellar Granule Cells J. Neurosci., March 1, 1998; 18(5): 1693 - 1703. [Abstract] [Full Text] [PDF]

				R. Mäkelä, M. Uusi-Oukari, G. E. Homanics, J. J. Quinlan, L. L. Firestone, W. Wisden, and E. R. Korpi Cerebellar gamma -Aminobutyric Acid Type A Receptors: Pharmacological Subtypes Revealed by Mutant Mouse Lines Mol. Pharmacol., September 1, 1997; 52(3): 380 - 388. [Abstract] [Full Text]

				S. Bahn, A. Jones, and W. Wisden Directing gene expression to cerebellar granule cells using gamma -aminobutyric acid type A receptor alpha 6 subunit transgenes PNAS, August 19, 1997; 94(17): 9417 - 9421. [Abstract] [Full Text] [PDF]

				V. Tretter, B. Hauer, Z. Nusser, R. M. Mihalek, H. Hoger, G. E. Homanics, P. Somogyi, and W. Sieghart Targeted Disruption of the GABAA Receptor delta Subunit Gene Leads to an Up-regulation of gamma 2 Subunit-containing Receptors in Cerebellar Granule Cells J. Biol. Chem., March 23, 2001; 276(13): 10532 - 10538. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (188) Citing Articles via Google Scholar Google Scholar Articles by Jones, A. Articles by Wisden, W. Search for Related Content PubMed PubMed Citation Articles by Jones, A. Articles by Wisden, W.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help