Article Figures & Data
Figures
- Figure 1.
α1-subunit-containing GABAARs are intimately associated with gephyrin. A, Lysates from 18–21 Div neurons were immunoprecipitated with anti-α1 or IgG. Precipitated material was immunoblotted with gephyrin (top) or anti-α1 (bottom) antibodies. B, A total of 2.5 μg of the respective fusion proteins was overlaid with 35S-gephyrin. The top shows an autoradiograph, and the bottom shows Coomassie staining of the same gel. The level of gephyrin binding was corrected for input and normalized to the level seen for the α1 subunit (α1 = 100%). Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001 (unpaired t test; n = 3).
- Figure 2.
Residues 360–375 within the α1 subunit mediate high-affinity binding to gephyrin. A, Fusion proteins encoding distinct portions of the intracellular domain of the α1 subunit were overlaid with 35S-gephyrin. Left, The top shows an autoradiograph, and the bottom shows Coomassie staining of the same gel. The level of gephyrin binding was corrected for input and normalized to the level seen for the α1 subunit (α1 = 100%). FL, Full length M3–M4 loop; NT, N-terminal half (334–375); CT, C-terminal half (376–419); Δ1, deletion 1 (360–375); Δ2, deletion 2 (384–395); α1, GST fusion protein. B, Alignment of homologous regions of α1 subunits of residues 360–375 of the α1 subunit. C, Overlay assays were performed using GST-α1 and GST-α1/6, and data were normalized to the level of binding seen for α1 (100%). Data represent mean ± SEM. ***p < 0.001 (unpaired t test; n = 3). D, Binding affinities were determined in ITC experiments by titrating the gephyrin E-domain (309–750, P1 variant) with α1 or α1/6 under similar experimental conditions. The measured binding enthalpies are plotted as a function of the molar ratio of the respective fusion proteins. Heat release over time is shown in the inset together with the average KD and its SD. N.D., Not detectable.
- Figure 3.
Preventing gephyrin binding to the α1 subunit disrupts GABAAR synaptic clustering. A, Nucleofected neurons (18–21 DIV) expressing GABAARs incorporating pHα1 and pHα1/6 (left) or pHα1Δ1 and pHα1Δ2 subunits (right) were fixed, permeabilized, and stained with antibodies against gephyrin (red) and VIAAT (blue). Neurons were visualized via confocal microscopy, and the right panels represent enlargements of the boxed areas in the large panels. Arrowheads denote clusters. B, HEK-293 cells expressing pHα1, pHα1/β2, pHα1/6/β2, pHα1Δ1/β2, or pHα1Δ2/β2 were fixed and stained with GFP antibodies without membrane permeabilization. Images were collected via confocal microscopy. Scale bars: 15 μm.
- Figure 4.
Preventing gephyrin binding to the α1 subunit decreases mIPSC amplitude. A, B, Sample traces are shown of mIPSCs recorded from 12 DIV neurons expressing either pHα1Δ1 or pHα1Δ2 subunits (A) or those expressing pHα1 or pHα1/6 subunits (B). These data were used to calculate mIPSC amplitude, frequency, rise, and decay times as detailed in the text.
- Figure 5.
Thr375 regulates the affinity of the α1 subunit for gephyrin. A, Alignment of residues 360–377 of the α1 and α2 subunits. Thr375 is highlighted in blue. Yeast transformants expressing gephyrin or control vector together with the intracellular domains of GlyR β or variants of α1 were assayed for β-gal activity. B, GST-α1, α1T375A, α1T375D, and α1T375E were subjected to SDS-PAGE and overlaid with 35S-gephyrin. Binding was visualized via autoradiography (left, top) and Coomassie staining (left, bottom). The level of gephyrin binding was then normalized to that seen for GST-α1 (100%; right). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01 (unpaired t test; n = 3). C, Binding affinities of gephyrin for α1 (■), α1T375A (□), and α1T375E (▴) as determined by ITC and representative titration curves are shown together with the average KD values and their SDs.
- Figure 6.
Thr375 regulates both the clustering of GABAARs at inhibitory synapses and mIPSC amplitudes. A, Nucleofected neurons (18–21 DIV) expressing GABAARs incorporating pHα1 or pHα1T375D subunits were fixed, permeabilized, and stained with antibodies against gephyrin (red) and VIAAT (blue). Neurons were visualized via confocal microscopy and the right panels represent enlargements of the boxed areas at left with arrowheads pointing to clusters. Synaptic clustering of pHα1T375D was severely attenuated compared to pHα1-subunit-expressing neurons (for details, see text). B, Typical traces of mIPSCs recorded from 12 DIV hippocampal neurons expressing pHα1 and pHα1T375D subunits. These data were then used to calculate mIPSC amplitude, frequency, rise, and decay times as detailed in the text.
- Figure 7.
Analysis of the diffusion of α1 and α1/6 subunits using single-particle tracking. Hippocampal neurons were cotransfected with either pHα1 or pHα1/6 subunits and gephyrin-mRFP at 10 DIV. Subunits were tracked at 21 DIV with anti-GFP-coupled QDs. Inhibitory synapses were identified by the presence of cotransfected gephyrin-mRFP. A1, A2, Examples of trajectories of pHα1 subunits in living neurons. A1, Codetection of pHα1 and gephyrin-mRFP. QD-coupled pHα1 subunits (blue dots) were visualized inside (yellow arrow) or outside (blue arrow) synapses (red). A2, Individual trajectories reconstructed from QDs shown in A1. Note that extrasynaptic subunits explored larger areas and moved faster (higher diffusion coefficients D) than synaptic ones. Scale bars: 1 μm. B, The cumulative distribution of diffusion coefficients of the α1- and α1/6-subunit-containing GABAARs showing the increased mobility of α1/6-type receptors at synapses. *p < 0.05 (Kolmogorov–Smirnov test). Inset, No difference could be seen outside synapses. C, Average MSD of α1 and α1/6 GABAARs at synapses. D, Comparison of surface areas explored by α1- and α1/6-subunit-containing GABAARs at synapses. QD trajectories were pooled according to their diffusion coefficients from the slowest (first) to the fastest (fourth) quartile. Note that the first and second lowest quartile groups exhibit a major increase for α1/6 compared to α1, and that this difference gradually decreases for faster subunits (first–third quartiles, ***p < 0.001; fourth quartile, not significant; t test). E, Receptor dwell times at synapses. Note the longer dwell times for α1 than for α1/6 subunits. Insets, Distribution of square distances (steps) covered by QDs for 1 s time intervals along the trajectories. Distribution of steps for QDs dwelling for <5 s at synapses (left inset) or >5 s at synapses (right inset). Note the major shift toward greater displacements for long-dwelling α1/6 subunits.
Tables
- Table 1.
Analysis of the binding of gephyrin to the intracellular domains of GlyR β and α1 subunit intracellular domains
Receptor loop (M3–M4) Stoichiometry (N) Affinity (in μm) Enthalpy (in kcal/mol) Measurements GlyR β WTa 0.65 ± 0.01 0.14 ± 0.08 −16 ± 1 1 0.60 ± 0.2 7.7 ± 0.1 −7 ± 2 GABAAR α1 WTb 0.62 ± 0.07 17 ± 11 −6 ± 7 11 GABAAR α1/6 chimerab N.D. N.D. N.D. 2 GABAAR α1 T375Eb 0.69 ± 0.001 183 ± 33 −6 ± 8 2 GABAAR α1 T375Ab 0.64 ± 0.028 36 ± 11 −15 ± 15 2 -
Purified fusion proteins encoding the intracellular domains of GlyR and GABAAR subunits were exposed to the residues 309–750 of the P1 variant of gephyrin, and the interaction was measured using ITC. All values represent mean ± SD; n = 11 for wild type (WT) and n = 2 for mutants. N.D., Not detectable.
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↵aAnalyzed with a two-site binding model. Top numbers refer to the high-affinity binding site. Data were taken from the study by Kim et al. (2006) for a representative measurement with SDs derived from the curve-fitting procedure in Origin.
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↵bAnalyzed with a single-site binding model. Mean values were calculated for several α1 measurements and are given with their SDs.
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