Edited by: Dorota Frydecka, Wroclaw Medical University, Poland
Reviewed by: Avishek Adhikari, Columbia University, USA; Malgorzata Maria Kossut, Nencki Institute of Experimental Biology, Poland
*Correspondence: Gert Lubec
†These authors have contributed equally to this work.
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GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM) task, a robust paradigm for evaluation of spatial learning. Trained or untrained (yoked control) C57BL/6J male mice (
Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system and mediates its effect through two distinct classes of GABA receptors: the ionotropic (GABAA and GABAC) receptors, which, when activated produce a rapid and very short-lived inhibition via chloride currents (Watanabe et al.,
GABAB receptors are obligatory heterodimers composed of GABAB1 and GABAB2 subunits, which are required for agonist binding and G-protein coupling (Robbins et al.,
Both, GABAB1a and GABAB1b receptor subunits are expressed in the hippocampus (Liang et al.,
A substantial body of evidence exists indicating the modulatory role of GABAB receptors in learning and memory. Stimulation of GABAB receptors impaired working memory in the basal forebrain (DeSousa et al.,
Crosstalk among the neurotransmitter repertoire is common in the central nervous system. This is made possible through formation of signaling complexes as a result of receptor assembly with other receptors or regulatory proteins. As studies so far have not been addressing GABAB receptor containing complexes as actors in the MTM, it was the aim of the current study to show which GABAB-containing receptor complex(es) are involved in a MTM task, a paradigm for evaluation of spatial learning.
C57BL/6J (
MTM is one of the spatial learning tasks in which animals learn to find the goal box based on their memory of previously visited arms and carried out as described elsewhere (Patil et al.,
A total of 20 samples (obtained from 10 trained and 10 untrained mice) were homogenized in ice-cold homogenization buffer (10 mM HEPES, pH 7.5, 300 mM sucrose and 1 complete protease inhibitor tablet [Roche Molecular Biochemicals, Mannheim, Germany] per 50 mL) using an Ultra-Turrax homogenizer (IKA, Staufen, Germany). The homogenate was then centrifuged for 10 min at 1000 × g, and the pellet was discarded. The supernatant was subsequently centrifuged at 50,000 × g for 30 min in an ultracentrifuge (Beckman Coulter Optima L-90K), and the resulting pellet was homogenized in 5 mL washing buffer (homogenization buffer without sucrose), incubated on ice for 30 min and centrifuged at 50,000 × g for an additional 30 min (Falsafi et al.,
The plasma membrane purification procedures for the obtained pellets were performed as previously described with slight modifications (Ghafari et al.,
Membrane pellets obtained from the 41% fraction of sucrose gradient ultracentrifugation were solubilized in extraction buffer [1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0] and 10% Triton X-100 stock solution was added, at a ratio of 1:4, to achieve a final concentration of 2% Triton X-100, with vortexing every 10 min for 1 h. Following solubilization, samples were cleared by centrifugation at 4°C and 20,000 × g for 60 min. The protein content was estimated using the BCA protein assay kit (Pierce, Rockford, IL, USA).
Following mixing of 100 μL of the membrane protein preparation with 16 μL BN-PAGE loading buffer [5% (w/v) Coomassie G250 in 750 mM 6-aminocaproic acid], 50 μg of the membrane protein preparations were loaded onto gels. BN-PAGE was performed in a PROTEAN II xi Cell (BioRad, Germany) using 4% stacking and 5–18% separating gels. The BN-PAGE gel buffer contained 500 mM 6-aminocaproic acid, 50 mM Bis–Tris, pH 7.0; the cathode buffer contained 50 mM Tricine, 15 mM Bis–Tris, 0.05% (w/v) Coomassie G250, pH 7.0; and the anode buffer contained 50 mM Bis–Tris, pH 7.0. The voltage was set to 50 V for 1 h followed by 75 V for 6 h, and then sequentially increased to 400 V (maximum current 15 mA/gel, maximum voltage 500 V) until the dye front reached to the bottom of the gel (Ghafari et al.,
Membrane proteins were transferred from BN-PAGE to PVDF membranes. After blocking of the membranes for 1 h with 10% non-fat dry milk in 0.1% TBST (100 mM Tris–HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), mouse anti-GABAB1 (Abcam, ab55051, Cambridge, UK; 1/5000). Goat anti-GABAB1a (sc-73401/2500), goat anti-MuscarinicM2 (Abcam, ab140473, Cambridge, UK; 1/3000) and rabbit anti-GABAB2 (Abcam, ab75838, Cambridge, UK; 1/5000). The antibodies were detected using horseradish peroxidase-conjugated secondary anti-rabbit IgG antibodies (Abcam, ab6721, Cambridge, UK; 1/10,000), anti-Goat IgG antibodies (Abcam, ab6741, Cambridge, UK; 1/5000), Anti-Mouse IgG antibodies (Abcam, 97035, Cambridge, UK; 1/10,000) Membranes were then developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). All membranes were stained with Coomassie blue R-350 (GE Healthcare, Buckinghamshire, UK, Falsafi et al.,
Total hippocampal membrane fractions were suspended in lysis buffer containing 1% Triton X100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0), 10 mM NaF, 10 mM Na3VO4 and protease inhibitor cocktail (Roche, Mannheim, Germany) on a rotating shaker for 1 h at 4°C. After centrifugation at 15,300 × g and 4°C for 10 min, the supernatant was incubated with affinity purified rabbit antibody against GABAB1 and subsequently incubated with protein G agarose beads (GE Healthcare, Uppsala, Sweden) for 4 h at 4°C with gentle rotation. After washing five times with the same lysis buffer, proteins bound were denatured with sample buffer containing 125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, and 0.02% bromophenol blue at 95°C for 3 min. Samples were then loaded onto 10% SDS-polyacrylamide gels, electrophoresed and subsequently transferred to PVDF membranes and analyzed by the western blotting as described in the previous subsection (Ghafari et al.,
Bands that were recognized by the corresponding antibodies against the GABAB1 receptor subunit were picked from the SDS gel and put into a 1.5 mL tube. Gel pieces were initially washed with 50 mM ammonium bicarbonate and then two times with washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) each time for 30 min with vortexing. An aliquot of 100 μL of 100% acetonitrile was added to the tubes to cover the gel pieces completely and the mixture was incubated for 10 min. Gel pieces were dried using a SpeedVac concentrator. Reduction of cysteine residues was carried out with 10 mM dithiothreitol (DTT) solution in 100 mM ammonium bicarbonate (pH 8.6) for 60 min at 56°C. After discarding the DTT solution, the same volume of 55 mM iodoacetamide (IAA) solution in 100 mM ammonium bicarbonate buffer (pH 8.6) was added and incubated in darkness for 45 min at 25°C to achieve alkylation of cysteine residues. The IAA solution was replaced by washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) and washed twice for 15 min each time with vortexing. Gel pieces were washed and dried in 100% acetonitrile followed by dryness in a SpeedVac. Dried gel pieces were re-swollen with 12.5 ng/μL trypsin (Promega, Germany) solution reconstituted with 25 mM ammonium bicarbonate. Gel pieces were incubated for 16 h (overnight) at 37°C (trypsin). The supernatant was transferred to new 0.5 mL tubes, and peptides were extracted with 50 μL of 0.5% formic acid / 20% acetonitrile for 20 min in a sonication bath. This step was repeated two times. Samples in extraction buffer were pooled in 0.5 mL tubes and evaporated in a SpeedVac concentrator. The volume was reduced to approximately 20 μL and an equal volume of HPLC grade water (Sigma, Germany) was then added for spectrometric analysis (Ghafari et al.,
The analysis utilized an Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA) equipped with a PepMap100 C-18 trap column (300 μm × 5 mm) and a PepMap100 C-18 analytic column (75 μm × 150 mm). The gradient applied was as follows: A = 0.1% FA in water and B = 0.08% FA in acetonitrile: 4–30% B from 0 to 105 min, 80% B from 105 to 110 min, and 4% B from 110 to 125 min. An HCT ultra ETD II (Bruker Daltonics, Bremen, Germany) was used to record the peptide spectra over a mass range of m/z 350–1500 and MS/MS spectra via information-dependent data acquisition over a mass range of m/z 100–2800. The MS spectra were repeatedly recorded, followed by four data-dependent CID MS/MS spectra and four ETD MS/MS spectra generated from the four highest intensity precursor ions. Active exclusion of 0.4 min following 2 spectra was used to detect low-abundance peptides. The voltage between the ion spray tip and spray shield was set to 1500 V. Drying nitrogen gas was heated to 150°C, and the flow rate was set to 10 L/min. The collision energy was set automatically according to the mass and the charge state of the peptides chosen for fragmentation. Multiple charged peptides were chosen for the MS/MS experiments due to their good fragmentation characteristics. The obtained MS/MS spectra were interpreted, and peak lists were generated with DataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany).
MASCOT searches against the most recent version of the UniProtKB database were performed using MASCOT 2.2.06 (Matrix Science, London, UK) for protein identification. The search parameters were set as follows: enzymes selected as trypsin with a maximum of 2 missing cleavage sites; species taxonomy limited to mouse; a mass tolerance of 0.2 Da for peptide tolerance, and 0.2 Da for MS/MS tolerance; an ion score cutoff below 15; fixed modification of carbamidomethyl (C); and variable modification of oxidation (M), deamidation (N, Q), and phosphorylation (S, T, Y). Proteins were positively identified based on a significant MOWSE score. Following protein identification, an error-tolerant search was performed to detect non-specific cleavage and unassigned modifications. The returned protein identification information was manually inspected and filtered to confirm the protein identification lists.
Higher sequence coverage was obtained using Modiro® software with the following parameters: the selected enzymes exhibited two maximum missing cleavage sites; a peptide mass tolerance of 0.2 Da was selected for peptide tolerance; 0.2 Da was used as the fragment mass tolerance; and modification 1 of carbamidomethyl (C) and modification 2 of methionine oxidation were applied. Positive protein identification was first based on the obtained spectra, and each identified peptide was subsequently considered significant based on its ion charge status, b- and y-ion fragmentation quality, ion score (>200) and significance scores (>80). The protein identifications were manually inspected and filtered to obtain the confirmed lists of identified proteins (Ghafari et al.,
For the antibody shift assay (ASA), samples were pre-incubated for an hour with mouse anti-GABAB1 (Abcam, ab55051, Cambridge, UK; 1/5000) and rabbit anti-GABAB2 (Abcam, ab75838, Cambridge, UK; 1/5000), with gentle shaking at 4°C, followed by loading the mixture onto the BN gel. The lanes from the BN gel were cut out and the gel strips were equilibrated for 30 min in equilibration buffer [1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol] with gentle shaking and then briefly rinsed twice with SDS-PAGE electrophoresis buffer [25 mM Tris–HCl, 192 mM glycine and 0.1% (w/v) SDS; pH 8.3] and subsequently by Milli-Q water. SDS-PAGE was performed in a PROTEAN II xi Cell using 4% stacking gel and 10% separating gel. The gel strips were run at 12°C with an initial current of 50 V (during the 1st h). The voltage was subsequently increased to 100 V for the next 12 h (overnight) and then increased to 150 V until the dye front reached the bottom of the gel. The gel was then further subjected to a western blotting analysis as described earlier.
Three C57BL/6J mice were anesthetized with 0.3 ml/kg intraperitoneal injection of sodium penthobarbital (Release, 300 mg/ml, Wirtschaftsgenossenschaft deutscher Tierärzte eG, Grabsen, Germany) and perfused intracardially with ice-cold PBS (0.1M phosphate buffered saline, pH 7.2), containing 0.2% heparin followed by 4% paraformaldehyde at a pH of 7.4 in PBS. Brain samples were post-fixed in 4% paraformaldehyde for 24 h at 4°C then transferred into a 30% sucrose solution (in PBS) for 48 h. Brain tissues were embedded with Tissue-Tek media (OCT compound, Sakura Finetek Europe, The Netherlands) then immersed in isopentane cooled with dry ice and sectioned at 30 μm with a cryostat (Leica CM 3050S, Wetzlar, Germany).
The Proximity Ligation Assay (PLA) was performed according to the manufacturer's protocol (O-LINK Bioscience, Uppsala, Sweden) with slight modifications. Free floating brain slices were blocked for 30 min at room temperature using blocking buffer supplied with the kit. After blocking, brain slices were incubated with diluted mouse monoclonal anti-GABAB1 (1:50, Abcam, Cambridge, UK) and rabbit polyclonal anti-GABAB2 receptor (1:25 Abcam, Cambridge, UK) primary antibodies for 72 h at 4°C on a rocking platform. Following incubation with primary antibodies, slices were washed with wash buffer A (O-LINK Bioscience, Uppsala, Sweden) and then incubated with rabbit PLUS and mouse MINUS probes (1:40, O-LINK Bioscience, Uppsala, Sweden) for 2 h at 37°C with gentle orbital shaking. Ligation was performed according to the manufacturer's protocol with the exception of the incubation time (45 min were used instead of 30 min mentioned in the protocol). DNA polymerase was diluted (1:20) and incubated for 120 min at 37°C. The rest of the amplification steps remained unchanged. After amplification, slices were washed with 1 × then 0.01 × wash buffer B prepared according to the recipe supplied in the kit manual. Finally, brain slices were transferred to glass slides, mounted with Duolink
Data obtained from MTM and western blotting are presented as mean ± SD. Data from western blot were analyzed by unpaired Student's
Mice learned the maze quickly and efficiently Schematic of the Multiple T maze (MTM) (Figure
To assess the role of GABAB receptors in the MTM task, exstirpated hippocampi from trained and untrained mice were subjected to blue native-PAGE followed by western blotting (BN-PAGE-WB). The analysis revealed the presence of a complex containing GABAB1 and GABAB2 subunits at an apparent molecular weight of approximately 600 kDa (Figure
Assays used for analysis of proteins showed the presence of the two subunits within the complex. The ASA is often used to verify the co-existence of a protein in a complex. The binding of specific antibodies to a target protein causes a shift of the whole target protein complex on the BN-PAGE by the added mass of the antibody. A corresponding mass shift of a protein complex, therefore, is indicative of the presence of the target protein in the complex. In this assay, antibodies specific for B1 and B2 subunits shifted the complex to higher apparent molecular weights, with the discrete increments most likely reflecting assembly of these subunits into GABAB receptors.
ASA was performed to provide additional evidence for co-assembly of the two subunits in order to form functional receptors (Figure
Immunoprecipitation was carried out using an antibody against the GABAB1 subunit (also used for Western blotting, see above) and the subunit was shown to co-precipitate with GABAB2. the GABAB1 receptor subunit was detected at an apparent molecular weight of approximately 100 kDa (Figure
GABAB1 | 960 | 108 | 23 | |
GABAB2 | 940 | 105 | 17 | |
CaMKII Alpha | 478 | 54 | 21 | |
CaMKII Beta | 542 | 60 | 12 | |
GRIA1 | 907 | 101 | 3 | |
GRIA2 | 883 | 98 | 10 |
Evidence for the presence of the two GABAB subunits within the complex was provided by the PLA (Figure
Spatial learning and memory is largely dependent on the hippocampus and is mediated by persistent changes in neuronal structure and activity. GABAB receptors are localized in the perisynaptic areas of dendritic spines as well as in the dendritic shafts, which enable them to regulate synaptic inputs (Bettler and Tiao,
The use of BN-PAGE allows subsequent immunoblotting of strongly hydrophobic membrane proteins as receptors and native receptor complexes including high molecular weight material (Kang et al.,
Most of the evidence relating GABAB receptors to learning and memory came out of studies using pharmacological ligands. Accordingly, antagonists appear to enhance cognition in majority of the cases (Bianchi and Panerai,
Subunit phosphorylation appears to regulate the surface dynamics of GABAB receptors. Phosphorylation at S867 by calcium/calmodulin-dependent protein kinase II (CaMKII) triggers endocytosis (Guetg et al.,
Mice with S783A mutation displayed significant alterations in contextual fear learning and reduced long-term spatial memory in Barnes's maze, effects thought to result from enhanced expression of postsynaptic GABAB receptors (Terunuma et al.,
The maximal distance between the secondary antibodies in PLA is slightly larger than the distance between flurophores in energy transfer technologies, allowing validation of the molecular proximity of endogenous proteins
It is known that the GABAB receptor is a heterodimer receptor, but so far mass spectrometric evidence for a complex containing the subunits from mouse brain is missing. Immunoprecipitation followed by mass spectrometry produced evidence for the presence of a protein complex containing both subunits along with proteins involved in the glutamatergic signaling system. Methodologically, nano-LC–ESI–MS/MS was applied for identification of receptor subunits and unambiguous identification with high sequence coverage was obtained. This was achieved through in-gel digestion with trypsin and the use of two mass spectrometric principles, the combination of CID and ETD.
The co-precipitation of the GABAB1 receptor with proteins involved in glutamatergic signaling indicates that the two systems interact with each other through formation of a signaling complex. Indeed, previous co-immunoprecipitation followed by pull down assay showed that CaMKII associates with the GABAB1 receptor in mouse brain. This association may enable CaMKII to phosphorylate GABAB receptor at S867 and trigger endocytosis (Guetg et al.,
Taken together, data presented herein demonstrate that training leads to increased levels of GABAB1/1a—containing receptor complexes but not GABAB2—containing receptor complexes in mouse hippocampus. The presence of these subunits in the complex is unequivocally confirmed using several biochemical methods including BN-PAGE, ASA, PLA, and immunoprecipitation followed by mass spectrometry. The findings are relevant for previous studies and the design of future studies on GABAB-receptors and spatial memory.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are highly indebted to Sabine Rauscher, Core SERD for excellent technical support of the study.
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