Compartmentalization of the GABAB receptor signaling complex is required for presynaptic inhibition at hippocampal synapses

Abstract

Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca(2+) channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 Å) complexes between GABA(B) receptors composed of GB(1a)/GB(2) subunits, Gα(o)β(1)γ(2) G-protein heterotrimer, and Ca(V)2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB(1a) deletion disrupted intermolecular associations within the complex. The GB(1a) proximal C-terminal domain was essential for association of the receptor, Ca(V)2.2 and Gβγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca(2+) transients and synaptic vesicle release. Thus, compartmentalization of the GABA(B1a) receptor, Gβγ, and Ca(V)2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.

Figure 1.

GB_1aRs, Gα_oβ₁γ₂ G-protein subunits, and Ca_V2.2 channels are precoupled at single hippocampal boutons. A, Representative confocal images of pyramidal neuron axons in hippocampal cultures that were cotransfected with GB_1a^YFP/Ca_V2.2^CFP, GB_1a^YFP/Gα_o^CFP, GB_1a^CFP/Gβ₁^YFP and Ca_V2.2^CFP/Gβ₁^YFP. Scale bars, 2 μm. B, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 33, N = 7), GB_1a^YFP/Gα_o*^94CFP (n = 53), GB_1a^CFP/Gβ₁^YFP (n = 45, N = 10), GB_1a^CFP/Gγ₂^YFP (n = 21, N = 4), Ca_V2.2^CFP/Gβ₁^YFP (n = 31, N = 6) proteins under miniature synaptic activity at single hippocampal boutons. To verify FRET specificity, E was measured between the CFP-tagged proteins of interest and nonrelated TNFR₂^YFP. Error bars indicate SEM. ***p < 0.001. C, FRET efficiency is plotted for individual presynaptic boutons as function of CFP/YFP intensity ratio (F_CFP/F_YFP). No correlation was found: Spearman r is 0.14, 0.11, −0.08, and 0.05 for GB_1a^YFP/Ca_V2.2^CFP, GB_1a^YFP/Gα_o*^94CFP, GB_1a^CFP/Gβ₁^YFP, and Ca_V2.2^CFP/Gβ₁^YFP, respectively (p > 0.5). D, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 8, N = 3), GB_1a^YFP/Gα_o^CFP (n = 9, N = 3), GB_1a^CFP/Gβ₁^YFP (n = 8, N = 3), and Ca_V2.2^CFP/Gβ₁^YFP (n = 12, N = 4) proteins in nonreleasing TeTx-pretreated hippocampal boutons. E, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 9, N = 3), GB_1a^YFP/Gα_o^CFP (n = 9, N = 3), GB_1a^CFP/Gβ₁^YFP (n = 10, N = 3), and Ca_V2.2^CFP/Gβ₁^YFP (n = 9, N = 3) proteins in nonreleasing immature (4–5 DIV) hippocampal neurons.

Figure 2.

Agonist-induced structural rearrangements in the GB_1aR/G-protein/Ca_V2.2 channel complex. A, E between GB_1a^YFP/Ca_V2.2^CFP was reduced by baclofen (10 μm, left, n = 13–29, N = 4–6, ***p < 0.0001), but was increased by CGP54626 (1 μm, right, n = 14, N = 4, **p < 0.01). B, E between GB_1a^YFP/Gα_o^CFP was reduced by baclofen (10 μm, left, n = 12–17, N = 4, ***p < 0.0001), but was increased by GABA_BR antagonist CGP35348 (1 μm, right, n = 13–18, N = 4, ***p < 0.0001). C, E between GB_1a^CFP/Gβ₁^YFP was increased by baclofen (10 μm, left, n = 12–15, N = 4, ***p < 0.0001), but was decreased by GABA_BR antagonist CGP54626 (1 μm, right, n = 15, N = 4, ***p < 0.0001). D, E between Gβ₁^YFP/Ca_V2.2^CFP was increased by baclofen (10 μm, left, n = 14–27, N = 4–6, ***p < 0.0001), but was decreased by CGP54626 (1 μm, right, n = 14–17, N = 4–5, ***p < 0.0001). Error bars indicate SEM. E, Fluorescence intensity of pHluorin tagged to GB_1a does not change under miniature activity, by application of 10 μm baclofen and as function of stimulation frequency (10 and 100 Hz). Slope of linear fit is 1.04, 0.98, 1.01, and 0.99 for miniature activity, baclofen application, 10 and 100 Hz, respectively.

Figure 3.

Disruption of FRET between the receptor, Ca_V2.2 channel, and Gβγ at hippocampal boutons of 1a^−/− neurons. A, Confocal images of axonal part of 1a^−/− hippocampal pyramidal neuron that was cotransfected with Ca_V2.2^CFP and Gβ₁^YFP. Scale bar, 2 μm. B, Lack of specific FRET between Ca_V2.2^CFP and Gβ₁^N′-YFP in 1a^−/− boutons: under miniature synaptic activity (Cnt, n = 31, N = 6), in the presence of 10 μm baclofen (n = 16, N = 4), and in TeTx-treated (n = 14, N = 3) and young (n = 14, N = 4) neurons. Transfection of 1a^−/− neurons with GB_1a resulted in rescue of Ca_V2.2^CFP/Gβ₁^N′-YFP FRET (n = 17, N = 4, ***p < 0.0001). C, Lack of specific FRET between Ca_V2.2^CFP and either Gγ₂^N′-YFP (n = 10, N = 3) or Gγ₂^C′-YFP (n = 9, N = 3). D, Disruption of specific FRET between Ca_V2.2^CFP and GB₂^YFP protein in 1a^−/− neurons (n = 8, N = 3, ***p < 0.0001). E, Diagram illustrating disruption of Ca_V2.2^CFP/Gβ₁^YFP FRET in boutons of 1a^−/− neurons. One-way ANOVA analysis with post hoc Bonferroni's multiple comparison tests (B) and paired t test (C, D) indicated significance. Error bars indicate SEM.

Figure 4.

Proximal C-terminal domain of the GB_1a protein is essential for Gβγ/Ca_V2.2 channel association. A, Schematics show GB_1a constructs used to examine the domain responsible for Gβγ/Ca_V2.2 association. SD, Two sushi domains; LBD, ligand-binding domain; 7TM, seven-transmembrane domain; PCT, proximal C-terminal domain; CC, coiled–coiled domain; DCT, distal C-terminal domain. B, Mean FRET for the indicated transfection conditions in 1a^−/− neurons: GB_1a-WT (n = 66, N = 11), GB_1a-Δ103 (n = 44, N = 8, **p < 0.01), GB_1a-Δ21 (n = 24, N = 6, **p < 0.0001), GB_1a-Δ74 (n = 14, N = 4, p > 0.05), GB_1a-Δ39 (n = 17, N = 4, p > 0.05), GB_1a-ΔSD (n = 15, N = 4, p > 0.05), and GB_1a-S269A (n = 14, N = 4, p > 0.05). One-way ANOVA analysis with post hoc Dunnett's multiple-comparison tests relative to 1a^−/− boutons transfected with GB_1a-WT indicated significance. C, Effect of 10 μm baclofen on the GB_1a^YFP/Ca_V2.2^CFP FRET in 1a^−/− boutons transfected with GB_1a-WT (n = 7–20, N = 3–5, **p < 0.01) or GB_1a-Δ21 (n = 23–24, N = 5–6, p > 0.05). D, Effect of 10 μm baclofen on the GB_1a^YFP/Gα_o^CFP FRET in 1a^−/− boutons transfected with GB_1a-WT (n = 12–15, N = 3–4, *p < 0.05) or GB_1a-Δ21 (n = 14–16, N = 3–5, *p < 0.05, paired t test). Error bars indicate SEM.

Figure 5.

The GB_1a proximal C-terminal domain does not affect baclofen-induced GB_1a/GB₂ receptor activation and cAMP inhibition. A, Dose–response curves of baclofen on GB_1a^CFP/GB₂^YFP FRET efficiency for GB_1a-WT (n = 10–21, N = 5, ED₅₀ = 0.82 ± 0.003 μm) and GB_1a-Δ21 (n = 11–21, N = 3–5, ED₅₀ = 0.58 ± 0.013 μm) proteins. E at 100 μm baclofen was set as 100%. B, Effect of 10 μm baclofen on CFP–Epac–YFP FRET efficiency (E_Epac), reporting cAMP level, in 1a^−/− boutons and in 1a^−/− boutons expressing GB_1a-WT or GB_1a-Δ21 proteins. Baclofen increased E_Epac in GB_1a-WT-expressing (n = 30–32, N = 6, *p < 0.05) and GB_1a-Δ21-expressing (n = 27–35, N = 5–6, *p < 0.05) boutons, but did not affect E_Epac in 1a^−/− boutons (n = 25–26, N = 5, p > 0.05) One-way ANOVA with post hoc Bonferroni's multiple-comparison tests indicated significance. Error bars indicate SEM.

Figure 6.

The role of the GB_1a proximal C-terminal domain on baclofen-induced inhibition of Ca²⁺ transients. A, Baclofen did not affect spike-dependent presynaptic Ca²⁺ transients (ΔF/F) evoked by 0.2 Hz stimulation in 1a^−/− boutons and in GB_1a-Δ21-expressing boutons, but reduced it in GB_1a-WT-expressing boutons. Ca²⁺ transients were quantified as before (black) and after (gray) baclofen application (average of 10 traces). B, Average data on baclofen-induced modification in Ca²⁺ transients in 1a^−/− (n = 31, N = 6, p > 0.05), GB_1a-WT-expressing (n = 17, N = 4, ***p < 0.001, compared with 1a^−/−), and GB_1a-Δ21-expressing (n = 19, N = 5, p > 0.05, compared with 1a^−/−) boutons. One-way ANOVA with post hoc Bonferroni's multiple-comparison tests indicated significance. Error bars indicate SEM.

Figure 7.

The GB_1a proximal C-terminal domain is required for baclofen-induced inhibition of synaptic vesicle release. A–E, Representative FM destaining curves before and after application of 10 μm baclofen in WT cultures (n = 84, A), WT cultures transfected with MyrPhd and GFP (n = 112, B), 1a^−/− cultures (n = 71, C), 1a^−/− cultures transfected with GB_1a-WT and GFP (n = 69, D), and 1a^−/− cultures transfected with GB_1a-Δ21 and GFP (n = 94, E). F, Summary of baclofen effect on FM destaining rate in WT (N = 12), GFP-expressing boutons (N = 3), WT cultures transfected with MyrPhd and GFP (N = 4), 1a^−/− (N = 9), 1a^−/− transfected with GB_1a-WT and GFP (N = 6), and 1a^−/− transfected with GB_1a-Δ21 and GFP (N = 6) cultures. Error bars indicate SEM. (***p < 0.001, n.s. for p > 0.05, one-way ANOVA analysis with post hoc Bonferroni's multiple-comparison tests).