Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex

Abstract

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.

Fig. 1

GABA_ARs are present in various subcellular brain fractions. The P2, SYN, SPM, PSD, were prepared as described in Materials and Methods. A–F show EM immunogold of synaptosomal (A–C) and “One Triton PSD” (D–F) fractions with rabbit anti-α1 GABA_AR subunit (A and D, arrowheads), mAb to gephyrin (B, C and E, arrowheads) and mAb to PSD-95 (F, arrowhead). Filled arrows indicate type-I PSDs, which have high electron-density. Empty arrows point to structures of medium electron-density immunolabeled with antibodies to α1 or gephyrin, likely corresponding to type-II PSDs. The secondary goat anti-rabbit IgG and anti-mouse IgG antibodies were conjugated to 6 nm and 12 nm diameter colloidal gold particles respectively. Scale bar = 100 nm. G: Immunoblots of various brain fractions with rabbit anti-α1, rabbit anti-α2, rabbit anti-γ2, and rabbit anti-gephyrin antisera, mAb to β2/3 and mAb to PSD-95. Same amount of total protein from each fraction (10μg/lane) was used for SDS-PAGE. The immunoreactivity of the specific protein band was blocked by the corresponding antigenic peptide (data not shown). H: [³H]MUS and [³H]FNZ binding to various brain fractions. Specific activity values (pmol/mg protein) are mean ± S.E.M of at least three experiments each done in triplicate. *** p < 0.001; ** p < 0.01. The p values are for the various fractions compared to PSD.

Fig. 2

In the “One Triton PSD” fraction, some GABA_ARs are associated with DRMs and some are associated with GABAergic type-II PSDs. Fractionation of “One Triton PSD” after centrifugation on a continuous 0.32–2.0 M sucrose density gradient. A: Distribution of cholesterol and protein in the density gradient. B: Immunoblots of the various fractions with antibodies to GABA_ARs subunits (α1, α2, α3, β2, β3, γ2, δ), Gephyrin, PSD-95, transferrin receptor (TrsfR) and the lipid raft markers Thy-1, Caveolin-1 and Flotillin-1. The same amount of total protein (4μg/lane) was used for SDS-PAGE. Fractions 1 and 12 correspond to the top and bottom of the gradient respectively. C: Double-labeled EM Immunogold of pooled gradient fractions 3 and 4 with rabbit anti-α1 and mAb anti-β2/3. Crossed arrows show the DRM vesicles present in this fraction. Some of these structures are immunolabeled with rabbit anti-α1 (filled arrowhead) and/or mAb anti-β2/3 (empty arrowheads). D–F: Double-label EM immunogold of fraction 7 with rabbit anti-γ2 and mAb to gephyrin. Filled arrow in F indicates a type-I PSD that has high electron-density. Empty arrows in D–F point to amorphous structures of medium electron-density. Some of these structures are immunolabeled with rabbit anti-γ2 (filled arrowheads) and/or mAb to gephyrin (empty arrowheads). Crossed arrows in D and E indicate vesicular membranes. The secondary goat anti-rabbit IgG and goat anti-mouse IgG antibodies were conjugated to 18 nm and 10 nm diameter colloidal gold particles respectively. Scale bar represents 150 nm in C and D and 100 nm in E and F.

Fig. 3

Some GABA_AR in synaptosomal plasma membrane are associated with DRMs. The SPM fraction was extracted by 1% Triton X-100 at 4°C and the soluble (TS) and insoluble (TI) fractions were collected. The low-density DRM fraction was also collected after flotation in a discontinuous sucrose gradient. A: Immunoblots with rabbit antibodies to α1, α2, β2, β3, γ2 GABA_AR subunits, gephyrin, transferrin receptor (TrsfR) and lipid raft markers Thy-1, Caveolin-1 and Flotillin-1. Same amount of total protein (4μg/lane) was used for SDS-PAGE. B: [³H]MUS and [³H]FNZ binding to GABA_ARs in TS, TI and DRM. Specific activity values (pmol/mg protein) are mean ± S.E.M of at least three experiments each done in triplicate. *** p < 0.001; ** p < 0.01. The p values are for the TS or TI fractions compared to DRM.

Fig. 4

Saponin treatment disrupts the association of GABA_ARs with DRMs. The SPM fraction was incubated with 0.5% saponin (S) or without saponin (C, control) followed by extraction with 1% Triton X-100 at 4°C, flotation in a sucrose density gradient and fractionation. Immunoblotting was done with rabbit antibodies to α1, β3, γ2 GABA_ARs, gephyrin, transferrin receptor (TrsfR) and the lipid raft markers Thy-1, Flotillin-1 and Caveolin-1. The same volume (12μl/lane) from each fraction was used for SDS-PAGE. Fractions 1 and 12 correspond to the top and bottom of the gradient respectively.

Fig. 5

In cultured hippocampal neurons some α1-GABA_AR clusters, but no γ2-GABA_AR clusters or gephyrin clusters, are associated with lipid rafts. Double-label immunofluorescence of pyramidal cell dendrites. Live hippocampal neurons were subjected to one-step capping with anti-γ2 or anti-α1 GABA_AR antibody and double-labeling with mAb to gephyrin or Thy-1 respectively. A–D: After one-step capping with guinea pig anti-γ2 (A and B) or rabbit anti-α1 (C and D), cells were fixed followed by incubation with Triton X-100 at 4°C (Triton X-100 after fixation, TX-AF). E–H: After surface labeling and one-step capping with guinea pig anti-γ2 (E and F) or rabbit anti-α1 (G and H), cells were treated with Triton X-100 at 4°C before fixation (TX-BF). I–L: After surface labeling and one-step capping with guinea pig anti-γ2 (I and J) or rabbit anti-α1 (K and L), cells were treated with MβCD before Triton X-100 extraction at 4°C followed by fixation (MβCD). Double-label immunofluorescence of γ2-GABA_AR subunit (A, E and I) and gephyrin (B, F and J) or α1-GABA_AR subunit (C, G and K) and Thy-1 (D, H and L). The secondary antibodies were Texas Red conjugated anti- guinea pig or anti- rabbit IgG and FITC conjugated anti- mouse IgG, all made in donkey. Scale bar: 10 μm. M and N: Quantification of cluster density and cluster size respectively. Data in M and N were collected from 30–40 dendrites from 15–20 cells of 3–4 separate experiments. *** p < 0.001; ** p < 0.01. The p values are for the TX-BF or MβCD compared to TX-AF.

Fig. 6

An anti-α1 antibody induces the capping of α1-GABA_ARs that are associated with lipid rafts. Double-label immunofluorescence of pyramidal cell dendrites. A–C: Hippocampal cultures were fixed, permeabilized with Triton X-100 and double-labeled with rabbit anti-α1 and mAb to gephyrin (no capping, TX-AF). D–L: Live neurons were one-step capped with anti-α1 antibody and double-labeled with mAb to gephyrin. M–U: Live neurons were two-step capped with sequential incubation of anti-α1 antibody and secondary antibody and then double-labeled with mAb to gephyrin. In TX-AF, one-step capping (D–F) or two-step capping (M–O) was followed by 0.25% Triton X-100 permeabilization after fixation. In TX-BF, one-step capping (G–I) or two-step capping (P–R) was followed by incubation with 0.5% Triton X-100 at 4°C before fixation. In MβCD, one-step capping (J–L) or two-step capping (S–U) was followed by treatment with MβCD before incubation with Triton X-100 at 4°C and fixation. Panels A–U show representative dendrites. Scale bar: 10 μm. V and W: Quantification of α1-GABA_AR and gephyrin cluster density and their co-localization in hippocampal cultures in the various experimental conditions represented in panels A–U. For quantification, 30 dendrites were randomly chosen from 15–20 cells from 3 separate experiments. Values represent mean ± SEM. *** p < 0.001; * p < 0.05.