SUPPLEMENTAL METHODS
RNA isolation and quantitative analyses of Gabra3 editing/expression
RNA was isolated and purified from mouse brain using Versagene RNA tissue and DNase kits (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. Unless otherwise specified, all brain tissues were isolated from 129S6 (12-
14 week) or FVB/J (12 week) male mice. Adult rat whole brain RNA (Sprague-Dawley) was isolated as previously described (Burns et al., 1997) and human whole brain RNA was purchased from BioChain Institute, Inc. (Hayward, CA). Complementary DNA was generated from 2 µg of total RNA using random priming with AMV reverse transcriptase
(Promega, Madison, WI). PCR amplification was performed with specific sense and antisense primers designed from GenBank sequences (Gabra1, NM_010250; Gabra2,
NM_008066; Gabra3, NM_008067; Gabra4, NM_010251; Gabra5, NM_176942;
Gabra6, NM_008068) to generate amplicons corresponding to the entire region of the predicted I/M site duplex within Gabra3 transcripts or the corresponding region for other
GABAA subunit isoforms (Supplementary Table 1). Bulk sequence analysis of PCR products was performed with an internal primer using BigDye Terminator chemistry and an ABI 3730xl analyzer (Applied Biosystems).
To quantify the extent of editing in mouse brain RNA, RT-PCR products were subcloned into pBKSII+ (Stratagene, La Jolla, CA) and DNA was extracted by heat lysis for use as the template for a custom-designed TaqMan SNP genotyping assay
(Supplementary Table 1; Applied Biosystems). Post-run analysis was performed with an
ABI Prism 7900HT system (SDS v2.3; Applied Biosystems). For quantitative analysis of Gabra3 mRNA expression, total RNA from whole mouse brain or dissected brain regions was reverse transcribed with random primers and the generated cDNAs were used as templates in a TaqMan MGB gene expression assay for Gabra3 (Mm00433440_m1) with 18S rRNA as an endogenous control (product no.
4319413E) from Applied Biosystems. Real-time PCR and subsequent analyses were performed using an ABI prism 7900HT (SDS v2.3; Applied Biosystems), and all reactions were run in triplicate and quantified as described previously (Feng et al., 2006).
Minigene construction and analysis
A wild-type Gabra3 minigene (543 bp), extending from a region in exon 6 through exon 9, was constructed by subcloning a non-edited Gabra3 amplicon generated from E15 mouse brain RNA into the pEGFP-C1 expression plasmid (Invitrogen,
Carlsbad, CA). Mutagenesis was performed by PCR with Pfu Ultra (Stratagene, La Jolla,
CA) in the presence of 5% DMSO. Template DNA was digested with DpnI (New
England Biolabs, Ipswich, MA) and the PCR product was transformed into DH5α- competent cells; all constructs were verified by DNA sequence analysis.
60-mm plates of HeLa cells, cultured in AMEM with 10% (v/v) FBS, were cotransfected using FuGENE 6 (Roche, Indianapolis, IN) with 0.5 µg of a Gabra3 minigene along with 4µg of either an empty pRC-CMV expression vector (Promega,
Madison, WI) or pRC-CMV containing the full-length cDNA encoding rat ADAR1
(p150) or rat ADAR2b, each with a FLAG epitope tag on the 5’-end. Cells were harvested 36 hours after transfection, RNA was isolated and purified using Versagene
RNA cell and DNase kits (Gentra Systems) and whole cell extracts were prepared from a parallel sample for Western blotting analysis, as described previously (Schreiber et al.,
1989). Western blotting analysis to quantify ADAR1/ADAR2 expression was performed using an anti-FLAG M2 monoclonal antibody (1:1000 v/v, Sigma-Aldrich, F3165) and an anti-β-tubulin polyclonal antibody (1:1000 v/v; Santa Cruz Biotechnology, sc-9104), detected with an Alexa Fluor 680-conjugated anti-rabbit (Molecular Probes, A21109) or
IRDye800-conjugated anti-mouse secondary antibody (Rockland, 610-732-124), respectively. Fluorescence was quantified using an Odyssey infrared imaging system (LI-
COR Biotechnology).
Analysis of editing was quantified by bulk sequence analysis of RT-PCR amplicons, generated as described above, but using 0.5 mg of RNA template and a transcript-specific RT primer containing a unique tail that was used to prime the PCR reaction (Supplementary Table 2), as described previously (Rueter et al., 1995).
Quantification was performed directly from electropherogram traces using a method adapted from Kwok et al. (Kwok et al., 1994) demonstrating that relative nucleotide peak heights are maintained from trace-to-trace when comparing the same region of a DNA sequence. The analysis was performed by dividing the height of the A peak at the editing site (I/M site) in a minigene sequence by an average of the heights of the three preceding adenosine peaks to provide a height normalized to the signal of that specific trace. A parallel analysis was performed on a control PCR amplicon generated from a non-edited
Gabra3 plasmid. The percentage of non-edited RNA in the population was calculated by dividing the normalized A peak height from a transfected sample by the normalized height from the non-edited plasmid control sample.
Expression of recombinant GABAA receptors
Complementary DNAs encoding the human GABAA receptor α3(I), α3(M) and
γ2L subunits were individually subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA); cDNA encoding the human GABAA receptor β3 subunit was subcloned into pCMV6-XL5 (Origene, Rockville, MD). HEK293T cells were transfected using a calcium phosphate precipitation technique, as previously described (Lagrange et al., 2007). Cells were transfected with a total of 12 µg GABAA receptor subunit, 4 µg of each subunit plasmid (ratio 1:1:1) as well as 2 µg of pHook-1
(Invitrogen, Carlsbad, CA) so that immunomagnetic bead selection could be performed on day two. Following selection, the cells were plated on 35 mm dishes, and recordings were made on day three, approximately 18-36 h after selection.
For editing analysis, RNA was isolated from HEK293T cells 2 days post- transfection using TRI Reagent (Molecular Research Center) and RT-PCR and bulk sequence analysis was performed as described for Gabra3 minigene analysis.
Electrophysiological recording and drug application
Whole cell voltage-clamp recordings were performed on transiently transfected
HEK293T cells. Cells were bathed in an external solution consisting of (in mM): NaCl
142, CaCl2 1, KCl 8 MgCl2 6, Glucose 10, HEPES 10, (pH 7.4, ≈320 - 340 mOsm) throughout the duration of the experiment. All recordings were done at room temperature.
Glass micropipettes were formed from thin-walled borosilicate glass with a filament
(World Precision Instruments, Sarasota, FL) with a P2000 laser electrode puller (Sutter
Instruments, San Rafael, CA) and fire polished with a microforge (Narishige, East Meadow, NY). Microelectrodes used for lifted cell recording had resistances of 0.7- 1.6
MΩ when filled with an internal solution consisting of (in mM): KCl 153, MgCl2 1,
2+ HEPES 10, EGTA 5, Mg -ATP 2 (pH 7.3, ≈ 300 - 310 mOsm) to produce an ECl of approximately 0 mV.
Membrane voltages were clamped at –20 mV using an Axopatch 200A amplifier
(Axon Instruments, Foster City, CA) amplifier. GABA was applied to lifted cells with a fast drug application device in which a Warner SF-77B Perfusion Fast-Step (Warner
Instrument Corporation, Hamden, CT) motor was used to rapidly translate a multibarrel square glass pipette (Friedrich and Dimmock, Millville, NJ), resulting in open-tip solution exchange times consistently ≤ 800 µs and solution exchange time around lifted cells of ≈ 1 ms(Lagrange et al., 2007). To minimize the error induced by uncompensated series resistance error, all currents larger than 6 nA were excluded from kinetic analysis.
Considering the low access resistance (generally 2-3 MΩ) and small cells (membrane capacitance usually 8-15 pF), this cutoff amplitude was expected to keep the membrane potential error difference between the groups to a minimum (≤ 2-3 mV). We found no correlation between current amplitude and any of the reported kinetic parameters. To confirm these results, all kinetic data were independently analyzed using linear regression to assess the interaction between isoform, current amplitude, and kinetic parameters. All of these fits showed the rise time and deactivation rates to be dependent on the isoform.
Finally, an independent statistical analysis of electrophysiological data was done by pairing each current from non-edited receptor with an amplitude-matched (± 15%) partner in the edited receptor group, followed by a Wilcoxon matched pairs test (data not shown). For generation of concentration-response relationships, peak GABAA receptor currents evoked by randomly sequenced concentrations of GABA were recorded with at least 45 s of wash between each application. This time was empirically determined to be sufficient for complete recovery from desensitization (data not shown). The responses were normalized to the current elicited by 1 mM GABA after a prolonged wash in external solution during each sweep. Data were excluded if there was a greater than 15% rundown of the maximal response between sweeps.
Data analysis
Currents were low-pass filtered at 2 kHz, digitized at 5-10 kHz, and analyzed using the pClamp 9 software suite. For those cells with very small (<50 pA) currents, rise time, desensitization, and deactivation were not determined. Current amplitudes and 10-
90% rise times were measured using the Axon Instruments Clampfit 9 software package.
The activation rate was calculated as the inverse of the 10-90% rise time. The desensitization and deactivation time courses of GABAA receptor currents were fit using the Levenberg-Marquardt least squares method with up to six component exponential
(-t/τn) functions of the form: Σ ane + C where t is time, n is the best number of exponential components, an is the relative ampl0itude of the nth component, τn is the time constant of the nth component, and C is the residual current at the end of the GABA application.
Additional components were accepted only if they significantly improved the fit, as determined by an F-test automatically performed by the analysis software on the sum of squared residuals. The time course of deactivation was summarized as a weighted time constant, defined by the following equation: . Numerical data were expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism (San Diego, CA). Data were compared using a Mann-
Whitney test for pairs of data or a Kruskal-Wallis for multiple comparisons. Changes in editing or expression over development were tested using a generalized Spearman rank- order correlation. The normalized peak response to 5 or 10 ms GABA application was compared to the peak current during 4 s GABA applications, using a Wilcoxon signed rank test to compare to a hypothetical value of 1.0. Statistical significance was taken as p
≤ 0.05.
Supplemental Legends
Supplemental Figure 1 Sequence analysis of cDNAs encoding mouse GABAA receptor
α subunits.
Sequence electropherogram traces from whole mouse brain-derived cDNAs are presented for the Gabra3 editing (I/M site) and the corresponding region from the other mouse GABAA receptor α subunits. The positions of the predicted RNA duplex region formed by an imperfect inverted repeat (arrows) and the I/M editing site within GABRA3 transcripts (yellow box) are indicated.
Supplemental Figure 2 An extended RNA duplex structure is required for efficient editing of mouse Gabra3 transcripts (I/M site) by ADARs.
A, The predicted RNA duplex formed by an imperfect, inverted repeat within exon 9 is presented for wild-type mouse Gabra3 transcripts with the I/M editing site shown in yellow. Disruption of the duplex structure by the introduction of site-specific mutations (M1) or restoration of the predicted secondary structure by introduction of compensatory mutations (M2) are shown in red and green, respectively. B, Quantification of I/M site editing for wild-type (wt), mutant (M1) and compensatory (M1+M2) Gabra3 minigenes expressed in transiently transfected HEK293 cells in the presence or absence of ADAR1/ADAR2 (mean ± SEM, n = 4, *p ≤ 0.05).
Supplemental Table 1 Summary of GABA-evoked responses.
The pharmacokinetic properties of currents evoked using a rapid drug-delivery device to apply GABA to HEK293T cells transfected with non-edited α3(I)β3γ2L or edited
* ** *** α3(M)β3γ2L GABAA receptors. p≤0.05; p≤0.01, p≤ 0.001
Supplemental Table 2. Oligonucleotide primers used for PCR-based analyses of
GABAA receptor α subunit editing.
The introduction of specific mutations within Gabra3 minigene constructs or the introduction of restriction sites for subcloning purposes are indicated with lowercase letters.