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Transfection GABA Receptor GFP-Tagged Subunits Neurons HEK 293 Cells
OVERVIEW
We describe an approach that may allow to study changes in the γ-aminobutyric acid (GABAA)receptor distribution with development and pharmacological treatments in living neurons. We produced expression vectors containing chimeras of the green fluorescent protein (GFP) linked to the C terminus of GABAA receptors α1, γ2, or the δ subunits. Human embryonic kidney (HEK) 293 cells were successfully transfected with α1-GFP cDNAs together with β3 subunit as indicated by the formation of green fluorescent clusters of receptor subunits that colocalized with immunospecific staining for the α1 subunits and by whole-cell recordings of GABA-activated Cl- currents. Although the current density was lower in these cells, GABA, bicuculline, and ZnCl2 actions were unaltered. Similarly, transfection with cDNAs encoding for the γ2-GFP chimera together with α1β3 subunit cDNAs produced clusters of subunits and GABA-activated chloride currents that were insensitive to blockade by ZnCl2 and that were potentiated by zolpidem. Lastly, δ-GFP chimeras transfection in HEK cells produced receptor insensitive to the potentiation by the neurosteroid THDOC. We then successfully transfected primary cultures of neocortical and cerebellar neurons with these GABAA receptor subunits— GFP chimeras. We obtained evidence of elongated cluster formation in both cell types that matched well, although not completely, endogenous receptor clusters as indicated by β2/3 staining, and also partially corresponded to synaptophysin positive punctae indicating synaptic localization of transfected subunits. Electrophysiological recordings from transfected neurons indicated that functional GABAergic synapses were still maintained. This approach will allow to follow targeting and distribution of GABAA receptor clusters in living neurons during development in culture and in different experimental conditions.
BACKGROUND
Receptor protein dynamics and interactions in living cells can been studied with fluorescent microscopy, photobleaching, and resonance energy transfer. It is therefore essential to be able to identify strong ligands or selective antibodies to link to fluorescent probes to allow similar studies. An alternative approach takes advantage of the possibility to tag receptor proteins with a fluorescent protein. To this aim, the GFP from the jellyfish Aequorea victoria is beginning to be extensively used for this purpose. Indeed, GFP-tagged proteins, both in the cytosol and in the plasma membrane, have been studied recently (See References 6 and 7 for review). A particularly useful application of GFP tagging of protein is to study the localization, distribution, and dynamics of neurotransmitter receptors. An essential condition for this study however is that tagging does not alter binding or functional properties of the receptor. A GFP-tagged version of the Nmethyl- D-aspartate (NMDA) receptor subunit NR1 (12) has been transfected in mammalian HEK 293 cells and has been demonstrated to produce functional NMDA receptors when cotransfected with NR2A subunit. Functional integrity of GFP-conjugated glycine receptor channels has been recently reported (8). Similar findings have been shown for both α and β adrenergic receptors (2,3,10) and voltage- gated calcium channels (9). Ligand gated receptor channels are heterooligomeric proteins made by multiple subunit. In particular, GABAA receptors are pentameric structures that comprise an ion channel for chloride ion. These receptors, localized in postsynaptic membranes throughout the central nervous system are responsible for inhibitory synaptic transmission (See Reference 11 for review). Tagging of one of the subunit may allow to observe the formation of functional receptor complexes as demonstrated for NMDA receptors in HEK cells (12). Indeed, Connor et al. (5) have recently demonstrated the formation of functional GABA channels tagged with GFP on the surface of Xenopus oocytes. These receptors have maintained both binding and electrophysiological properties of nontagged receptors. Additionally, the level of receptor expression was unaltered, and both Ec50 for GABA and benzodiazepine potentiation were also unaffected. Lastly, the interaction between distinct subunit that gives rise to specific pharmacological properties was preserved when GFP-tagged α1 subunit is expressed. Calcium phosphate-based transient transfection technique (4) has been classically applied to transfect eukaryotic expression vectors in mammalian tumoral cell lines. Recently however, successful transfection of primary neuronal culture has been reported, although with a limited efficiency (1,16). Here, we report the successful construction of cDNAs expressing GABAA receptor subunits tethered to the GFP protein and its expression and colocalization in primary cultures of cortical and cerebellar neurons.
PROTOCOLS
Materials and Reagents
- Basal Eagle’s medium (Life
Technologies, Gaithersburg, MD, USA).
- Coverslips were mounted on slides
using Vectashield (Vector Laboratories, Burlingame, CA, USA) as mounting medium.
- Fluorescein isothiocyanate (FITC)-
conjugated goat antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
- Gentamycin (Life Technologies).
- Glass capillaries (Wiretrol II; DRUMMOND
Scientific, Broomall, PA, USA).
- Glass coverslips (Fisher Scientific, Pittsburgh, PA, USA).
- HEK 293 cells (No. CRL1573;
ATCC, Rockville, MD, USA).
- Indocarbocyanine (Cy3) (Jackson
ImmunoResearch Laboratories).
- Minimal essential medium (MEM)
(Life Technologies).
- MEM with Hanks’ salts (Life
Technologies).
- Monoclonal mouse antisynaptophysin
(Roche Molecular Biochemicals, Mannheim, Germany).
- Penicillin (Life Technologies).
- Plasmid pGREENLANTERN (Life
Technologies).
- Polyclonal rabbit anti-α1 and β2/3
antibodies (CHEMICON International, Temecula, CA, USA).
- Poly-L-lysine (Sigma, St. Louis, MO,
USA).
- Rabbit IgG antibodies (Jackson ImmunoResearch
Laboratories).
- Streptomycin (Life Technologies).
- Trypsin (Sigma).
- Vector pCDM8 (Invitrogen,
Carlsbad, CA, USA).
- Vector pEGFP-N1 (CLONTECH
Laboratories, Palo Alto, CA, USA).
- Patch amplifier (Axopatch 1D; Axon
Instruments, Foster City, CA, USA).
- 8-pole low-pass Bessel filter
(Frequency Devices, Haverhill, MA, USA).
- Digitization software (Clampex 8;
Axon Instruments).
- Analysis software (Origin; MicroCal
Software, Northampton, MA, USA and Clampfit 8; Axon Instruments).
Procedures
HEK 293 Cell Line HEK 293 cells were grown in MEM, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/ mL streptomycin, in a 6% CO2 incubator. Exponentially growing cells were dispersed with trypsin, seeded at 2 × 105 cells/35- mm dish in 1.5 mL of culture medium and plated on 12-mm glass coverslips. Primary Cultures Primary cultures of rat cerebellar granule neurons were prepared from 7 days old Sprague Dawley rat cerebella. Cortical neurons from newborn rat pups were prepared with similar procedure. Cells were dispersed with trypsin (0.25 μg/mL) and plated at a density of 0.8 to 1 × 106 on 35- mm Nunc dishes coated with poly-Llysine (10 μg/mL). Cells were cultured in basal Eagle’s medium supplemented with 10% bovine calf serum, 2 mM glutamine, 100 μg/mL gentamycin, and maintained at 37°C in 6% CO2. Cytosine arabinoside (10 μM) was added to all cultures 18 to 24 hours after plating to inhibit glial proliferation. The final concentration of KCl in the culture medium was adjusted to 25 mM.
Construction of Plasmid DNA for GABA Subunit with GFP Tags
Rat α1, γ2, and δGABAA receptor subunit cDNAs were individually subcloned into the expression vector pCDM8. To fuse those cDNAs with GFP, we used pEGFP-N1 vector that contains multiple cloning sites before the EGFP coding sequence. The GABAA receptor subunit cDNAs was modified via polymerase chain reaction (PCR), and the stop codons were replaced by suitable cloning site. Then the target gene was cloned into pEGFP-N1 so that it is in frame with the EGFP coding sequences. The α1 subunit was cloned into pEGFP-N1 using HindIII and KpnI sites. The PCR product of γ2 subunit was cut by BamHI and SpeI, and ligated to the pEGFP-N1 cut by NheI and BamHI. The δ subunit was inserted into EcoRI and BamHI sites of pEGFP-N1. cDNA Transient Transfection HEK 293 cells were transfected using the calcium phosphate precipitation method (4) with various combinations of subunit cDNAs. The following plasmid combinations were mixed: α1:β3:γ2-GFP, α1-GFP:β3:γ2, and α1:β3:δGFP, (1 μg each construct) and the coprecipitates were added to culture dishes containing 1.5 mL MEM for 12 to 16 hours at 37°C under 3% CO2. The media was removed, the cells were rinsed twice with culture media, and finally incubated in the same media for 24 hours at 37°C under 6% CO2. Cortical neurons and cerebellar granule cells at 4 days in vitro (DIV) were transfected with pEGFP-N1 expression vector, γ2-GFP, and α1-GFP chimera expression vector using a modified calcium phosphate precipitation method. Briefly, the culture medium was replaced and saved. After one time washing with transfection medium (MEM with Hanks’ salts), 3 μg plasmids were added to cells of each dish containing 1.5 mL transfection medium and then incubated for 1 hour at room temperature in cell culture hood. After rinsing 2 times with transfection medium, the cells were put back to saved medium and then were maintained at 37°C under 6% CO2. For α1-GFP chimera transfected cortical neurons, insulin was added to the medium at the concentration of 2 μm/mL in neurons at DIV 13 for 2 hours.
Electrophysiological Studies
Cultured granule cells or isolated HEK 293 transfected cells were voltage-clamped at -50 mV in the whole-cell configuration using the patch clamp technique on the stage of an inverted microscope at room temperature. The recording pipet contained (mM) 145 CsCl, 5 MgCl2, 11 ethylene glycol bis (β-aminoethlether)-N, N, N′,N′-tetraacetic acid (EGTA), 5 Naadenosine- 5′-triphosphate (ATP), 0.2 guanosine-5′-triphosphate (GTP), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.2 with CsOH. Cells were bathed in (mM) 145 NaCl, 5 KCl, 2 CaCl2, and 5 HEPES at pH 7.2 with NaOH. Osmolarity was adjusted to 325 mosm with sucrose. The culture dish in the recording chamber (< 500 μL total volume) was continuously perfused (5 mL/min) to prevent accumulation of drugs. All drugs dissolved in bath solution contained, dimethyl sulfoxide (DMSO) at a maximal final concentration of 0.01%, which failed per se to modify GABA responses. GABA was applied directly by a gravity-fed Y tubing delivery system placed within 100 μm of the recorded cell. Bath perfusion for the 2 minutes preceding coapplication with GABA was required to observe full potentiation of GABA responses by flunitrazepam. GABA (0.5 M in water adjusted to pH 4.0 with HCl) was also applied by iontophoresis with 30-millisecond pulses of positive current. With GABA iontophoretic currents in the 25 to 50 nA range, outward currents were generated in transfected cells in such a way as to obtain a peak amplitude of 150 to 200 pA. Benzodiazepines (BZs) were a gift from Hoffman La Roche (Switzerland), DMCM and β-CCM were from Ferrosan (Denmark) and Zolpidem was from Syntelabo (France). All drugs were dissolved in bath solution containing DMSO at a maximal final concentration of 0.01%.
Currents were monitored with a patch
amplifier (Axopatch 1D), filtered at 1.5
kHz (8-pole low-pass Bessel), digitized in
an IBM-PC computer with the Clampex 8
software for off-line analysis. After normaltionship
was performed using the logistic
equation:
% Imax = 100/Imax * {1+(EC50/[GABA]nh)}
where Imax is the maximal Cl- current,
elicited by GABA, EC50 is the GABA
concentration eliciting the half-maximal
response, and nh is the Hill coefficient.
Results are expressed as mean ± SEM.
Origin and Clampfit 8 software were used
for figure preparation and statistical analysis
using ANOVA with a P < 0.05 and a
paired t-test with P < 0.01. The Bonferroni
correction was applied for multiple group
comparison.
Antibodies
The monoclonal mouse anti-GAD65 antibody was a kind gift from Dr. Samuel Rabkin (Georgetown University, Washington, DC). Immunocytochemistry Cultured HEK 293 cells and were fixed with 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline (PBS) for 15 minutes at room temperature and permeabilized with 0.25% Triton X-100 for 3 to 5 minutes. Cells were preincubated in 10% goat serum for 30 minutes at room temperature and then incubated in primary antibody in PBS containing 5% goat serum overnight at 4°C. The concentrations of primary antibodies were: rabbit anti-β2/3 subunit (10 μg/mL), rabbit anti-α1 subunit (1:100), and mouse anti-GAD65 (1 μg/mL). After washing 3 times in PBS, cells were incubated with goat antirabbit and/or antimouse (Cy3-conjugated 1:200, FITCconjugated 1:50) secondary antibodies for 1 hour at room temperature. Coverslips were visualized using a microscope equipped for the visualization of fluorescence (Vanox, Olympus, Japan). Spectral characteristics of the excitation–emission filters used were 490/530 nm (green fluorescence for FITC and GFP) and 545/610 nm (red fluorescence for Cy3), respectively. Bleed-through was minimal as seen looking at immunostaining with only single color labeling and by the lack of colocalization of some clusters in double staining experiments. Cells were visualized through 10×, 40×, and 100× objectives, and images were captured using a digital camera and transferred to a computer workstation. Controls were performed by omitting primary antibodies. Only a weak and nonspecific staining was observed under these experimental conditions. Receptor cluster colocalization was quantified over a length of 100 μm in 3 dendrites evaluated in each of 5 selected neurons. Clusters were counted after background subtraction and manual fluorescent thresholding correspondent to twice the intensity of the diffuse fluorescence of the dendritic shaft
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