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• W2059097542 abstract "A GABAA receptor α1 subunit epilepsy mutation (α1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the α1 subunit. We reported previously that heterologous expression of α1(A322D)β2γ2 receptors in mammalian cells resulted in reduced total and surface α1 subunit protein. Here we demonstrate the mechanism of this reduction. Total α1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 ± 6%) or when coexpressed with β2 and γ2S subunits (78 ± 6%), indicating an expression reduction prior to subunit oligomerization. In α1β2γ2S receptors, endoglycosidase H deglycosylated only 26 ± 5% of α1 subunits, consistent with substantial protein maturation, but in α1(A322D)β2γ2S receptors, endoglycosidase H deglycosylated 91 ± 4% of α1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the α1 subunit was tagged with yellow (α1-YFP) or cyan (α1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 ± 4% of α1-YFPβ2γ2 but only 5 ± 1% α1(A322D)-YFPβ2γ2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 ± 4% α1-YFPβ2γ2S, 86 ± 3% α1(A322D)-YFP). Heterozygous expression of α1-CFPβ2γ2S and α1(A322D)-YFPβ2γ2S or α1-YFPβ2γ2S and α1(A322D)-CFPβ2γ2S receptors showed that membrane GABAA receptors contained primarily wild type α1 subunits. These data demonstrate that the A322D mutation reduces α1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane α1 subunits that are almost exclusively wild type subunits. A GABAA receptor α1 subunit epilepsy mutation (α1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the α1 subunit. We reported previously that heterologous expression of α1(A322D)β2γ2 receptors in mammalian cells resulted in reduced total and surface α1 subunit protein. Here we demonstrate the mechanism of this reduction. Total α1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 ± 6%) or when coexpressed with β2 and γ2S subunits (78 ± 6%), indicating an expression reduction prior to subunit oligomerization. In α1β2γ2S receptors, endoglycosidase H deglycosylated only 26 ± 5% of α1 subunits, consistent with substantial protein maturation, but in α1(A322D)β2γ2S receptors, endoglycosidase H deglycosylated 91 ± 4% of α1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the α1 subunit was tagged with yellow (α1-YFP) or cyan (α1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 ± 4% of α1-YFPβ2γ2 but only 5 ± 1% α1(A322D)-YFPβ2γ2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 ± 4% α1-YFPβ2γ2S, 86 ± 3% α1(A322D)-YFP). Heterozygous expression of α1-CFPβ2γ2S and α1(A322D)-YFPβ2γ2S or α1-YFPβ2γ2S and α1(A322D)-CFPβ2γ2S receptors showed that membrane GABAA receptors contained primarily wild type α1 subunits. These data demonstrate that the A322D mutation reduces α1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane α1 subunits that are almost exclusively wild type subunits. GABAA 2The abbreviations used are: GABA, γ-aminobutyric acid; AChR, nicotinic acetylcholine receptor; GABAA receptor, γ-aminobutyric acid receptor type A; M3, transmembrane domain 3; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; FP, fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; IDV, integrated density volume; endo-H, endoglycosidase H; PNGaseF, peptide N-glycosidase-F; WT, wild type; AFU, arbitrary fluorescence units. 2The abbreviations used are: GABA, γ-aminobutyric acid; AChR, nicotinic acetylcholine receptor; GABAA receptor, γ-aminobutyric acid receptor type A; M3, transmembrane domain 3; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; FP, fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; IDV, integrated density volume; endo-H, endoglycosidase H; PNGaseF, peptide N-glycosidase-F; WT, wild type; AFU, arbitrary fluorescence units. receptors are pentameric ligand-gated chloride ion channels that are the major inhibitory neurotransmitter receptors in the mammalian central nervous system (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1766) Google Scholar). The five subunits arise from seven subunit families that contain multiple subtypes and assemble in a limited number of subunit combinations, with the most prevalent consisting of two α1 subunits, two β2 subunits, and one γ2 subunit (2McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1069) Google Scholar, 3Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar, 4Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 5Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2002; 277: 46020-46025Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Each subunit contains four hydrophobic segments (M1-M4) that are homologous to the four membrane spanning helices of the Torpedo marmorata nicotinic acetylcholine receptor (AChR) subunits whose three-dimensional structure has been determined to 4 Å (6Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1071) Google Scholar). A nonconserved missense mutation in the GABAA receptor α1 subunit gene (GABRA1, α1(A322D)) that codes for an aspartate in place of an alanine at position 7 of the M3 transmembrane segment is present in a form of autosomal dominant juvenile myoclonic epilepsy (7Cossette P. Liu L. Brisebois K. Dong H. Lortie A. Vanasse M. Saint-Hilaire J.M. Carmant L. Verner A. Lu W.Y. Wang Y.T. Rouleau G.A. Nat. Genet. 2002; 31: 184-189Crossref PubMed Scopus (514) Google Scholar), an idiopathic generalized epilepsy syndrome that accounts for ∼10% of all cases of epilepsy (8Genton P. Gelisse P. Arch. Neurol. 2001; 58: 1487-1490Crossref PubMed Scopus (35) Google Scholar). When expressed in heterologous cells, this mutation affects both the function and expression of GABAA receptors. Expression of the α1(A322D) subunit with β2 and γ2 subunits (“homozygous expression”) reduced peak currents by ∼90%, substantially altered whole cell current kinetics, and reduced mean single channel open times (7Cossette P. Liu L. Brisebois K. Dong H. Lortie A. Vanasse M. Saint-Hilaire J.M. Carmant L. Verner A. Lu W.Y. Wang Y.T. Rouleau G.A. Nat. Genet. 2002; 31: 184-189Crossref PubMed Scopus (514) Google Scholar, 9Fisher J.L. Neuropharmacology. 2004; 46: 629-637Crossref PubMed Scopus (38) Google Scholar, 10Gallagher M.J. Song L. Arain F. Macdonald R.L. J. Neurosci. 2004; 24: 5570-5578Crossref PubMed Scopus (41) Google Scholar). We recently reported that the α1(A322D) mutation reduced α1 subunit expression by 94%, and that it produced asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and α1 subunit protein expression (10Gallagher M.J. Song L. Arain F. Macdonald R.L. J. Neurosci. 2004; 24: 5570-5578Crossref PubMed Scopus (41) Google Scholar). Heterozygous receptors constructed from concatamers with the α1(A322D) subunit positioned between two β2 subunits had 35% of peak current amplitudes and 70% of protein expression relative to wild type receptors, whereas heterozygous receptors with α1(A322D) positioned between β2 and γ2 subunits had 1% of peak current amplitude and 51% of protein expression of wild type receptors. To our knowledge, this is the first naturally occurring missense mutation that reduces expression of a ligand-gated ion channel subunit. Because α1(A322D) substantially reduced the amount of α1 subunit, it is likely that it is this reduction in total α1 subunit expression, and not the alteration of GABAA receptor current kinetics, that is the predominant mechanism by which this mutation causes disinhibition and epilepsy. Here we determined the mechanism by which this single missense mutation reduced α1 subunit expression. Expression of recombinant GABAA Receptors—pcDNA3.1 plasmids containing cDNAs that encode human α1, β2S, and γ2S GABAA receptor subunits were a gift from Dr. Mathew Jones (University of Wisconsin, Madison, WI). α1-YFP and α-CFP cDNAs were constructed by first inserting HpaI and SacII restriction sites between the codons encoding amino acids four and five of the mature α1 subunit. DNA encoding the fluorescent protein (FP) from the corresponding γ2S-FP subunit (11Kang J. Macdonald R.L. J. Neurosci. 2004; 24: 8672-8677Crossref PubMed Scopus (106) Google Scholar) was removed by HpaI and SacII digestion and then ligated into the α1 subunit-containing plasmid between the codons encoding amino acids four and five of the mature subunit. Cycle 3 GFP-tagged α1 subunit was constructed by performing a blunt ligation of the α1 subunit cDNA into cycle 3 GFP-containing pcDNA3.1 plasmid (Invitrogen). The α1(A322D) mutation was made using the QuikChange site-directed mutagenesis kit (Stratagene). All cDNA sequences were confirmed by DNA sequencing. Human embryonic kidney cells (HEK293T) were a gift from P. Connely (COR Therapeutics, San Francisco, CA). Cells were grown at 37 °C in 5% CO2, 95% air using Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Invitrogen) and 100 IU/ml streptomycin and penicillin (Invitrogen). Cells were transfected using the FuGENE 6 transfection reagent (Roche Diagnostics, 2.7 μl/μg of DNA). For the Western blot experiments, the cells were transfected in 6-cm dishes (Corning, Corning, NY) using 2:2:2 μg, α1:β2S:γ2S. Transfections were similar for the electrophysiology experiments but also included 1 μg of pHOOK (Invitrogen), which was used for immunomagnetic separation (12Greenfield L.J. Sun F. Neelands T.R. Burgard E.C. Donnelly J.L. Macdonald R.L. Neuropharmacology. 1997; 36 (Jr.): 63-73Crossref PubMed Scopus (49) Google Scholar). Twenty hours after transfection, cells were trypsinized, centrifuged (400 × g), and incubated with hapten-coated magnetic beads for 30 min at 37 °C. The cells that bound the magnetic beads were isolated via a magnetic stand and plated on 35-mm culture dishes. For the confocal microscopy experiments, cells were transfected in 35-mm dishes using equal amounts (1 μg) of α1-FP, β2S, and γ2S subunit cDNAs. For the ER colocalization experiments, the cells were also transfected with 30 ng of a cyan fluorescent protein-tagged endoplasmic reticulum marker (CFP-ER, BD Biosciences). Immunoblots—The Western blot protocol has been described (10Gallagher M.J. Song L. Arain F. Macdonald R.L. J. Neurosci. 2004; 24: 5570-5578Crossref PubMed Scopus (41) Google Scholar). Transfected cells were lysed in modified radioimmunoassay solution (RIPA, 20 mm Tris, pH 7.4, 1% Triton X-100, 150 mm NaCl, 0.25% deoxycholate) that contained one pellet of Complete Mini™ protease inhibitor (Roche Diagnostics) per 10 ml. The lysates were centrifuged at 10,000 × g for 30 min. Lysates were fractionated by SDS-PAGE at the acrylamide concentrations given in the figure legends. After SDS-PAGE, the proteins were electrotransfered to polyvinylidene fluoride membranes (Millipore Inc.). All primary antibodies were monoclonal and were purchased from Chemicon Inc. (Temecula, CA). In experiments in which the cells were transfected with untagged α1 and α1(A322D) subunits, the membranes were first incubated with an antibody against the α1 subunit (5 μg/ml, clone BD24) in addition to an antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 0.1 μg/ml, clone 6C5), which was used to control for the amount of protein loaded on the gel. For experiments in which the cells were transfected with FP-tagged α1 subunit, the membranes were incubated with a monoclonal antibody to green fluorescent protein (1:2500) as well as to an antibody to β-actin (clone C4, 1 μg/ml). After incubation with the primary antibody, all immunoblots were incubated with a horseradish peroxidase-coupled goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, 1:6000 dilution) and then visualized with a chemiluminescent detection system (Amersham Biosciences) using a digital imager (Alpha Innotech, San Leandro, CA). The integrated density volume (IDV, pixel intensity × mm2) of each band was calculated using the Alpha Innotech software. Background IDVs were obtained from regions adjacent to the bands of interest and subtracted from the total IDV. All protein bands were normalized to the loading control (GAPDH or actin). Endoglycosidase Digestion—Protein lysates were prepared as described above and their protein concentrations were measured using the Micro BCA Protein Assay™ (Pierce). Endoglycosidase H (endo-H) and peptide N-glycosidase-F (PNGaseF) were obtained from Sigma. Endo-H and PNGaseF digestion of nicotinic AChR δ-subunit has been described (13Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Both endoglycosidase digestions were performed for 3 h at 37 °C. Endo-H digestions were performed in 50 mm sodium citrate, pH 5.5, 1% Triton X-100, 0.1% SDS, 50 mm β-mercaptoethanol with 0.2 units/ml endo-H. PNGaseF digestions were performed in 50 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 50 mm β-mercaptoethanol with 0.2 units/ml PNGaseF. The reactions were terminated by addition of Laemmli sample buffer, and the reaction products were detected by SDS-PAGE and Western blot as described above. Confocal Microscopy and Image Analysis—Approximately 24 h before the confocal microscopy experiments, the transfected cells were plated in collagen-coated 35-mm glass-bottom dishes (MatTek, Ash-land, MA). The dishes were then coded, and thus the microscopy experiments were performed in a single-blinded fashion. Immediately before imaging, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4-64, 0.3 μg/ml, Invitrogen) was added to the culture media to stain the cells' plasma membranes without staining the ER (14Bolte S. Brown S. Satiat-Jeunemaitre B. J. Cell Sci. 2004; 117: 943-954Crossref PubMed Scopus (86) Google Scholar). Cells were chosen at random and were imaged using a Zeiss 500 META confocal microscope with a ×40, 1.3 numerical aperture Pan neofluoar objective. For all cells except those transfected with α1-CFP, the pinhole of all channels was adjusted so that the images were obtained as a single 2-μm slice through the middle of the cell. To collect more emitted light from the cells that were transfected with α1-CFP, the pinhole of all channels was adjusted so that the images were obtained as a single 3-μm slice through the middle of the cell. Excitation wavelengths were 455, 514, and 543 nm for CFP, YFP, and FM 4-64, respectively. For each excitation wavelength, the laser power was adjusted as necessary to utilize the full dynamic range of the detector for each respective fluorophore. CFP emission was detected with a 475-525-nm band pass filter, and FM 4-64 emission was detected using a 560-long pass filter. YFP emission was spectrally separated from CFP and FM 4-64 by reflecting the emitted light off a NFT545 dichroic mirror and then filtering it through a 530-600-band pass filter. The digital images were obtained with 2.8 times scanning zoom and 8 bit, 512 × 512 pixel resolution. All images presented in the figures are unprocessed. The confocal image files were coded and processed in a single-blind fashion using the ImageJ software (National Institute of Health, Bethesda, MD). Background FP fluorescence for each image was obtained from cells that stained with FM 4-64 but did not express FP. Background CFP-ER fluorescence was defined as the CFP fluorescence in the nucleus. The background fluorescence was subtracted from the images at the 99th percentile. For cells cotransfected with CFP-ER, membrane YFP fluorescence was defined as the YFP outside the boundary of CFP-ER and FM 4-64 staining. For cells not cotransfected with CFP-ER, membrane FP fluorescence was defined as the FP outside the inner portion of FM 4-64 staining. ER YFP fluorescence was defined as intracellular YFP that colocalized with ER fluorescence. The integrated fluorescence from each subcellular region was calculated by summing all the background-subtracted fluorescence values from each pixel in that area. Fluorescence Spectroscopy—Lysates of cells expressing β2 and γ2S subunits and wild type, heterozygous, or homozygous cycle 3 GPP-tagged α1 subunit (α1-GFP) were prepared as described above. Cycle 3 GFP was used to tag the α1 subunit for fluorescence spectroscopy rather than enhanced fluorescence proteins (CFP or YFP) because of its high fluorescence efficiency (15Crameri A. Whitehorn E.A. Tate E. Stemmer W.P. Nat. Biotechnol. 1996; 14: 315-319Crossref PubMed Scopus (1044) Google Scholar); it was not used for microscopy because its absorbance maximum is in the ultraviolet region of the spectrum. Ali-quots (200 μl) of the lysates were placed in microtiter plates, and the fluorescence was determined in a Flexstation™ fluorescence spectrometer (Molecular Devices, Sunnyvale, CA) with an excitation of 395 nm and emission of 507 nm. Background fluorescence was defined as the fluorescence from lysates from untransfected cells; the background was subtracted from the fluorescence of the experimental lysates. Electrophysiology—Electrophysiological recordings were performed 48 h after transfection. The intrapipette solution contained (in mm): 153 KCl, 1 MgCl2, 5 EGTA, 10 HEPES, and 2 MgATP (pH 7.3, osmolarity = 305-310 mOsm). The external recording solution consisted of (in mm): 142 NaCl, 8 KCl, 6 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES (pH 7.4, osmolarity = 319-325 mOsm). Recording pipettes were pulled on a Sutter P-2000 micropipette electrode puller (Sutter Instrument Co., San Rafael, CA) from borosilicate capillary glass (Fisher). GABA was applied to the cells via gravity using a system consisting of pulled four-barrel square glass (200-300 μm) connected to a Perfusion Fast-Step (Warner Instrument Corp., Hamden, CT). The solution exchange time was determined by stepping a 10% dilute external solution across the open electrode tip to measure a liquid junction current; the 10-90% rise times for solution exchange were consistently ≤0.4 ms. GABAA receptor currents were recorded in voltage clamp mode with cells clamped at -50 mV using a lifted whole cell patch-clamp technique (16Hinkle D.J. Bianchi M.T. Macdonald R.L. BioTechniques. 2003; 35 (476): 472-474PubMed Google Scholar) using an Axon 200B amplifier (Molecular Devices). The signals were sampled at 10 kHz and written to a computer hard drive. Homology Modeling and Secondary Structure Prediction—Multiple amino acid sequences were aligned using the ClustalW software (www.ebi.ac.uk/clustalw/). We identified amino acids of the M3 segments of the Torpedo marmorata AChR subunits that were homologous to the Ala-322 residue of the GABAA receptor α1 subunit in two steps. First, the following amino acid sequences were aligned in four groups: 1) Torpedo marmorata nAChR α, β, γ2, and δ subunits; 2) human GABAA receptor α1-6 subunits; 3) human GABAA receptor β1-3 subunits; and 4) human γ1-3 subunits. Next, the 16 sequences in these four groups were aligned together in conjunction with the human GABAA receptor δ-subunit, and the results of the 17-sequence alignment were visually compared with the small group analyses to make certain the sequences that were most similar with one another remained in alignment. Identification and scoring of transmembrane helices were performed using the relevant ExPASY Proteomics tools (us.expasy.org/tools/) topology prediction algorithms (DAS, TopPred, TMPred, and TMHMM). The predictions were performed first on the native AChR sequences and then on the sequences in which the amino acid in the M3 domain homologous to the GABAA α1 subunit Ala-322 was changed to an aspartate. The scores from the topology prediction programs, which predicted a transmembrane helix for the 25-amino acid segments beginning at αTyr-277, βTyr-283, γTyr-291, and δTyr-286 (corresponding to the M3 transmembrane helices in the Torpedo marmorata electron diffraction data) for the sequences with or without the aspartate substitution, were compared. Data Analysis—Values are reported as mean ± S.E. Statistical significance for the endoglycosidase, confocal, and fluorescence spectroscopy experiments were determined using the Student's unpaired t test and the significance of expression differences in the Western blot experiments and the effect of aspartate substitution on topology prediction scores were determined using the paired t test (GraphPad, San Diego, CA). α1(A322D) Subunit Expression Was Reduced With or Without Coexpression with β2 and γ2S Subunits—GABAA receptor subunits oligomerize in the ER via distinct pathways (17Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) utilizing specific intersubunit contacts (18Klausberger T. Sarto I. Ehya N. Fuchs K. Furtmuller R. Mayer B. Huck S. Sieghart W. J. Neurosci. 2001; 21: 9124-9133Crossref PubMed Google Scholar, 19Klausberger T. Ehya N. Fuchs K. Fuchs T. Ebert V. Sarto I. Sieghart W. J. Biol. Chem. 2001; 276: 16024-16032Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 20Klausberger T. Fuchs K. Mayer B. Ehya N. Sieghart W. J. Biol. Chem. 2000; 275: 8921-8928Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Bollan K. King D. Robertson L.A. Brown K. Taylor P.M. Moss S.J. Connolly C.N. J. Biol. Chem. 2003; 278: 4747-4755Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Taylor P.M. Thomas P. Gorrie G.H. Connolly C.N. Smart T.G. Moss S.J. J. Neurosci. 1999; 19: 6360-6371Crossref PubMed Google Scholar, 23Taylor P.M. Connolly C.N. Kittler J.T. Gorrie G.H. Hosie A. Smart T.G. Moss S.J. J. Neurosci. 2000; 20: 1297-1306Crossref PubMed Google Scholar), and unassembled subunits are degraded (24Gorrie G.H. Vallis Y. Stephenson A. Whitfield J. Browning B. Smart T.G. Moss S.J. J. Neurosci. 1997; 17: 6587-6596Crossref PubMed Google Scholar). Because expression of α1(A322D)β2γ2S receptors reduced currents and protein expression in a subunit position-dependent fashion (10Gallagher M.J. Song L. Arain F. Macdonald R.L. J. Neurosci. 2004; 24: 5570-5578Crossref PubMed Scopus (41) Google Scholar), we hypothesized that α1(A322D) subunit mutation inhibited subunit oligomerization, thereby leading to mutant α subunit degradation. To test this hypothesis, we transfected cells with wild type α1 or heterozygous or homozygous α1(A322D) subunits and either wild type β2 and γ2S subunits (α1β2γ2S) or an equivalent amount of empty pcDNA3.1 plasmid (α1pcDNA) and performed Western blots on whole cell lysates (Fig. 1, A and B). The ratio of α1 subunit to GAPDH expression from whole cell lysates was determined by quantification of Western blots from whole cell lysates (Fig. 1, C and D). With transfection of both α1β2γ2S and α1pcDNA subunits, heterozygous α1 subunit expression was intermediate between those of wild type and the homozygous mutant α1 subunit expression (percentage of wild type expression of α1β3γ2S: n = 10, heterozygous 66 ± 6%, p = 0.045, homozygous 22 ± 6% p = 0.011; α1pcDNA n = 10: heterozygous 49 ± 6%, p = 0.005, homozygous 14 ± 6%, p = 0.003). There were no significant differences in the reduction of α1 subunit expression between cells transfected with α1β2γ2S subunits and those transfected with α1 subunits alone (heterozygous p = 0.061, homozygous p = 0.335). These results were unexpected because they indicated that that the α1(A322D) mutation reduced α1 subunit protein levels prior to receptor assembly. α1(A322D) Was Endoglycosidase H Sensitive—A reduction in α1(A322D) subunit expression prior to receptor assembly could result from decreased efficiency of transcription or translation or from increased post-translational degradation of the mutant α1(A322D) subunit, known as ER-associated degradation (ERAD). If α1(A322D) subunit transcription or translation were reduced, the post-translational processing of residual α1(A322D) subunit would be similar to that of the wild type subunit. In contrast, accelerated ERAD would result in the residual α1(A322D) subunits having ER- but not Golgi-associated processing. The α1 subunit has two sites of N-linked glycosylation that reside on its extracellular N terminus (17Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 25Buller A.L. Hastings G.A. Kirkness E.F. Fraser C.M. Mol. Pharmacol. 1994; 46: 858-865PubMed Google Scholar). Membrane proteins are N-linked glycosylated with high mannose carbohydrates co-translationally within the ER, but upon trafficking to the trans-Golgi the high mannose carbohydrates are replaced with low mannose carbohydrates (26Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1578) Google Scholar). Digestion with endo-H removes high mannose N-linked carbohydrates, whereas digestion with PNGaseF removes all carbohydrates. Therefore, endo-H sensitivity indicates a protein has not been trafficked at least as far as the trans-Golgi (27Matsuda S. Hannen R. Matsuda K. Yamada N. Tubbs T. Yuzaki M. Eur. J. Neurosci. 2004; 19: 1683-1690Crossref PubMed Scopus (13) Google Scholar). To determine the glycosylation state of mutant α1(A322D) subunits, we digested cell lysates with endo-H or PNGaseF from cells transfected with wild type α1β2γ2S, heterozygous α1α1(A322D)β2γ2S, and homozygous α1(A322D)β2γ2S receptor subunits. Undigested cell lysates and those digested with endo-H or PNGaseF were analyzed by Western blot (Fig. 2, A and B). Because the wild type α1 subunit expresses more efficiently than heterozygous and homozygous α1 subunits, protein was loaded in the ratio WT:heterozygous: homozygous, 8:15:50 μg, to balance the amount of α1 subunit detected on the blot. Undigested wild type, heterozygous, and homozygous proteins ran at 50 kDa, and those digested with PNGaseF (removal of all N-linked carbohydrates) ran at 46 kDa, a reduction in molecular weight similar to PNGaseF digestion of Torpedo AChR δ-subunit (13Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and tunicamycin inhibition of GABAA receptor 9E10-tagged α1 subunit glycosylation (17Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). With endo-H digestion, wild type and heterozygous receptors ran in two bands at 46 (endo-H sensitive) and 48.4 kDa (endo-H resistant), whereas endo-H digestion products of homozygous receptors ran in a single band at 46 kDa (Fig. 2, C and D). The 1.6-kDa shift in the endo-H-resistant band is similar to endo-H digestion of the AChR δ-subunit (13Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Quantification of the 46- and 48.8-kDa digestion products demonstrated that 74 ± 5% of wild type (n = 6), 64 ± 4% of heterozygous (n = 6, p = 0.13), and 9 ± 4% (n = 6, p < 0.0001) of homozygous receptors were endo-H resistant. This shows that essentially all the homozygous α1(A322D) subunits retained ER processing and provides evidence that the defect in α1(A322D) subunit trafficking occurs after translation. Given the results of endo-H digestion of wild type and homozygous α1 subunits, with independent processing of these subunits, 42, not 64%, of heterozygous α1 subunit should have been endo-H sensitive. This suggests that other processes (such as protein degradation) may alter the relative ratios of wild type α1 and α1(A322D) subunits. Another alternative is that this assay lacks the resolution and statistical power to detect a difference in the extent of glycosylation between wild type and heterozygous receptors. α1-YFPβ2γ2S andα1(A322D)-YFPβ2γ2S Receptors Were Functional—To further characterize the subcellular localization of wild type and mutant α1 subunits, we constructed YFP-tagged wild type (α1-YFP) and mutant (α1(A322D)-YFP) subunits. Electrophysiological analysis of N-terminal fluorescent-tagged γ2-subunit-containing GABAA receptors has been performed in fibroblasts (11Kang J. Macdonald R.L. J. Neurosci. 2004; 24: 8672-8677Crossref PubMed Scopus (106) Google Scholar, 28Kittler J.T. Wang J. Connolly C.N. Vicini S. Smart T.G. Moss S.J. Mol. Cell. Neurosci. 2000; 16: 440-452Crossref PubMed Scopus (68) Google Scholar), and electrophysiological analysis of GABAA receptors containing α1 subunits tagged with GFP at the C terminus has been done in oocytes (29Connor J.X. Boileau A.J. Czajkowski C. J. Biol. Chem. 1998; 273: 28906-28911Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We evaluated GABA-evoked currents in cells transfected with GABAA receptors containing wild type or mutant α1 subunits tagged with YFP at the N terminus. Currents were evoked from both wild type α1-YF" @default.
• W2059097542 created "2016-06-24" @default.
• W2059097542 creator A5017109968 @default.
• W2059097542 creator A5033029238 @default.
• W2059097542 creator A5044198884 @default.
• W2059097542 creator A5060718314 @default.
• W2059097542 date "2005-11-01" @default.
• W2059097542 modified "2023-09-27" @default.
• W2059097542 title "Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit" @default.
• W2059097542 cites W1482147550 @default.
• W2059097542 cites W1482497049 @default.
• W2059097542 cites W1509483696 @default.
• W2059097542 cites W1575801138 @default.
• W2059097542 cites W1583705151 @default.
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• W2059097542 cites W2039451567 @default.
• W2059097542 cites W2047097502 @default.
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• W2059097542 cites W2051151140 @default.
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• W2059097542 cites W2077609671 @default.
• W2059097542 cites W2078720061 @default.
• W2059097542 cites W2087005030 @default.
• W2059097542 cites W2087823077 @default.
• W2059097542 cites W2090289021 @default.
• W2059097542 cites W2094885938 @default.
• W2059097542 cites W2096557041 @default.
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• W2059097542 cites W2138001567 @default.
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• W2059097542 cites W2152236836 @default.
• W2059097542 cites W2171381392 @default.
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