Fig 1: Interactions of EMC3 and EMC6 with neurotransmitter-gated ion channels(A) Co-immunoprecipitation (Co-IP) from primary rat cortical neurons demonstrated endogenous interactions between a1 subunits of GABAA receptors and EMC3, EMC6, and a number of a1-interacting chaperones (BiP and calnexin) and ERAD factors (Grp94 and VCP). Neurons were plated onto 10-cm dishes at a density of one million per dish. At DIV 12, proteins were extracted for Co-IP. IgG was used as a negative control during the immunoprecipitation. n = 3.(B) Co-IP from primary rat cortical neurons demonstrated endogenous interactions between EMC3/EMC6 and a number of ion channels, including N-methyl-D-aspartate receptors (NMDARs, including NR1, NR2A and NR2B subunits) and nicotinic acetylcholine receptors (nAChR a7 subunit). n = 3.(C) Co-IP from mouse cortical homogenates, which were prepared from C57BL/6J mice between 8 and 10 weeks of age, demonstrated endogenous interactions between EMC3/EMC6 and selected ion channels. n = 3.(D) Schematic of the primary sequence of EMC3 and EMC6. R31 and R180 in EMC3 and N22 and D27 in EMC6 were reported to influence the biogenesis of EMC-dependent client proteins.(E) Mutation of R31A or R180A in EMC3 significantly reduced the interaction of EMC3 with GABAA a1 subunits. The cDNAs of FLAG-tagged EMC3, either in the wild type (WT) form or carrying appropriate mutations of R31A or R180A, were transiently transfected in HEK293T cells stably expressing a1ß2?2 GABAA receptors. 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-FLAG M2 magnetic beads. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect a1 subunits and FLAG. Quantification of the band intensity of a1 over FLAG after immunoprecipitation was shown on the right (n = 3).(F) Mutation of D27A or N22A in EMC6 significantly reduced the interaction of EMC6 with GABAAR a1 subunits. Transfection of cDNAs was applied similarly as in E, however with co-application of FLAG-tagged EMC5 and EMC6 variants in HEK293T cells stably expressing a1ß2?2 GABAA receptors. Co-IP and visualization of protein bands were carried out the same way as in E as well. Quantification of the band intensity of a1 over FLAG-tagged EMC6 after immunoprecipitation was shown on the right (n = 3).(G) Significant increase of the interaction of SEC61a and a1 subunits of GABAA receptors was observed when both EMC3 and EMC6 were knocked down. We carried out siRNA transfection in HEK293T cells stably expressing a1ß2?2 GABAA receptors; 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-a1 antibody. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect SEC61a and a1 subunits. Quantification of the band intensity of SEC61a over a1 after immunoprecipitation was shown on the right (n = 3). Each data point is presented as mean ± SEM *, p< 0.05; **, p< 0.01. NT: Non-targeting scrambled siRNA; IP: immunoprecipitation; EV: empty vector; WT: wild type.
Fig 2: Effect of EMC3 and EMC6 on the protein levels and whole-cell patch-clamping currents of endogenous GABAAreceptors(A) Mouse GT1-7 neurons were incubated with siRNA against EMC3 or EMC6 for 48 h. Proteins were extracted and analyzed by western blotting; normalized band intensity was shown below the images (n = 3), with ß-actin as the loading control.(B) Mouse GT1-7 neurons were incubated with siRNA against EMC3 or EMC6 for 48 h. Whole-cell patch-clamping was performed on the cells with the IonFlux Mercury 16 ensemble plates at a holding potential of -60 mV. GABA (1 mM) was applied for 4 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by Fluxion Data Analyzer (n = 6 - 10). NT: Non-targeting scrambled siRNA; pA, picoampere.(C) Confocal microscopy imaging of primary rat cortical neurons demonstrated reduced surface expression of GABAA receptors after siRNA treatment of EMC3 and EMC6 through lentivirus transduction. Lentiviruses were generated from transiently transfected HEK293T cells with the following plasmids and collected after 60 h from the media passing through 0.45 µm filter: EMC3- or EMC6-set of four siRNA lentivectors, packaging and envelop plasmids. At day-in-vitro (DIV) 6 of the primary rat cortical neurons, lentivirus transduction was carried out at a multiplicity-of-infection (MOI) of 10. At DIV 12, neurons were stained for cell surface GABAA receptor a1 subunits (top row), ß2/ß3 subunits (middle row), and ?2 subunits (bottom row), colored in red. DAPI staining for the nucleus was colored in blue. Scale bar = 20 µm. Quantification of the fluorescence intensity by using ImageJ was shown on the bottom after background correction from 20–30 neurons. Each data point is presented as mean ± SEM *, p< 0.05; **, p< 0.01.
Fig 3: Overexpression of EMC3, and EMC5 and EMC6 restores surface expression and whole-cell currents of disease-associated variants of GABAA receptors(A–C). Overexpression of EMC3 and EMC5/6 increased surface expression of a1 subunits of GABAAR in HEK293T cells stably expressing a1(D219N)ß2?2 (A), a1(G251D)ß2?2 (B) and a1(P260L)ß2?2 (C). We carried out cDNA transfection of EMC3, or co-application of EMC5 and EMC6, in corresponding HEK293T cells; 48 h after transfection, surface proteins were enriched through biotin-neutravidin affinity purification, and western blot analysis was applied to detect a1 subunits. Na+/K+ ATPase served as loading control of cell surface proteins. Normalized surface a1 band intensity was shown below the images (n = 3).(D) HEK293T cells stably expressing a1(G251D)ß2?2 GABAA receptors were transfected with empty vector control (CTL) or EMC5 and EMC6 cDNAs. 48 h after transfection, cycloheximide (CHX) (100 µg/mL), a potent protein synthesis inhibitor, was added to cell culture media for the indicated time. Cells were then harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the a1 band intensity was plotted against the incubation time with CHX (n = 3).(E–G) Increased whole-cell patch-clamping currents of GABAA receptors were recorded in HEK293T cells stably expressing a1(D219N)ß2?2 (E), a1(G251D)ß2?2 (F) and a1(P260L)ß2?2 (G). Transfection of cDNA was applied the same way as in (A–C); 48 h after transfection, patch clamping was performed on the cells with the IonFlux Mercury 16 ensemble plates at a holding potential of -60 mV. GABA (100 µM) was applied for 4 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by Fluxion Data Analyzer (n = 6 - 10). Each data point is presented as mean ± SEM *, p< 0.05; **, p< 0.01. CTL: Empty vector control sample.
Fig 4: EMC3 and EMC6 promote anterograde trafficking of GABAA receptors(A and B) Significant reduction of cell surface and total a1 and ß2 subunits of GABAA receptors was observed when both EMC3 and EMC6 were knocked down. We carried out siRNA transfection in HEK293T cells stably expressing a1ß2?2 GABAA receptors. To test the surface expression of GABAA receptors, biotinylation experiments were performed 48 h after siRNA transfection of both EMC3 and EMC6 (A). Surface proteins were enriched through biotin-neutravidin affinity purification, and western blot analysis was applied to detect surface a1 and ß2 subunits. Na+/K+ ATPase served as loading control of cell surface proteins. To test the total protein expression of GABAA receptors, cells were lysed and total proteins were collected and subjected to SDS-PAGE and western blot analysis (B). ß-actin was used as the total protein loading control. Normalized band intensity was shown on the right to the blots (n = 3).(C) The ratio of the surface/total subunits of GABAA receptors was quantified, as a measure of their surface trafficking efficiency. Data was taken from (A) and (B) for the calculation.(D) HEK293T cells stably expressing a1ß2?2 GABAA receptors were transfected with non-targeting siRNA or siRNAs against EMC3 and EMC6. 48 h after transfection, cycloheximide (CHX) (100 µg/mL), a potent protein synthesis inhibitor, was added to cell culture media for the indicated time. Cells were then harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the a1 band intensity was plotted against the incubation time with CHX (n = 3).(E and F) EMC3 and EMC6 promote GABAA receptors’ trafficking from the ER to Golgi as demonstrated through endoglycosidase H (Endo H) digestion. EMC3 or EMC6 siRNA transfection was applied in HEK293T cells stably expressing a1ß2?2 GABAA receptors; 48 h after transfection, proteins were extracted, and subjected to Endo H digestion and western blot analysis. Endo H resistant bands (top two bands in lanes two and 4) represent proteins that have correctly folded in the ER, trafficked to Golgi and fully modified with the N-linked complex glycans, thus becoming resistant to Endo H. On the other hand, acting upon proteins remaining in ER, Endo H may remove the high mannose structure after the asparaginyl-N-acetyl-D-glucosamine on the a1 subunits, generating Endo H sensitive bands (bottom band in lanes two and 4). The Peptide-N-Glycosidase F (PNGase F) enzyme-treated samples served as a control for unglycosylated a1 subunits (lane 5). Quantification of the ratio of Endo H resistant/total a1 band intensity, as a measure of the trafficking efficiency of a1 subunits, was shown on the right (n = 3).(G) HEK293T cells stably expressing a1ß2?2 GABAA receptors were transfected with non-targeting siRNA or siRNAs against EMC3, EMC6, or both EMC3 and EMC6. 48 h after transfection, cells were harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the normalized individual EMC subunit band intensity was shown on the right panels (n = 3). Each data point is presented as mean ± SEM *, p< 0.05; **, p< 0.01. NT: Non-targeting scrambled siRNA.
Supplier Page from Thermo Fisher Scientific for EMC6 Antibody