Fig. 6.

Diazepam triggers dissociation of PLCδ from GABA_ARs in situ leading to activation of PLCδ/Ca²⁺/calcineurin signalling pathway, which is negatively regulated by PRIP1. a Immunoprecipitates of GABA_ARs from control and diazepam (D; 1 μM)-treated cortical neurones were probed with PLCδ- (n = 3) or b PRIP1-  (n = 4) specific antibody. c–e PLCδ-GFP binds directly to the intracellular loop of the GABA_AR β₂ and β₃ subunits Q1 (303-366 aa) and Q3 (366-396 aa) regions in GST pull-down assays (n = 3). f PRIP1-GFP binds directly to the β₃ subunit Q1 and Q3 loop regions in the GST pull-down assays (n = 3). g Predicted PLCδ- and PRIP1- binding sites in the Q1 and Q3 regions of the β₂ and β₃ subunits. h Immunoprecipitates of GABA_ARs from control (DMSO) or Isoguvacine (I; 5 μM)-, Diazepam (D; 1 μM)/Isoguvacine (I; 5 μM)- or Isoguvacine (I; 50 μM)-treated α₁β₂γ₂-GABA_AR HEK293 cells expressing both GFP-PLCδ and GFP-PRIP1 were probed with the GFP-specific antibody (n = 2). (i) Overexpression of PRIP1 inhibits partial translocation of GFP-PH_PLCδ1 in response to Diazepam (D; 1 μM)/Isoguvacine (I; 5 μM) in α₁β₂γ₂-HEK293 cells. Live imaging of a Calcein blue-labelled cell (blue) expressing GFP-PH_PLCδ1 (green) and dsRed-PRIP1 (red; top panels) and superimposed fluorescence intensity profiles prior to (left) and 5 min after the addition of Diazepam (D; 1 μM)/Isoguvacine (I; 5 μM) (right). j Quantification of fluorescence F(membrane)/F(cytoplasm) ratio of GFP-PH_PLCδ1 (green; left) and dsRed-PRIP1 (red; right), both shown as mean ± s.e.m. (Student t-test; *p < 0.05; n = 10 cells from 2 independent experiments). k Overexpression of PRIP1 inhibits Diazepam (D; 1 μM)/Isoguvacine (I; 5 μM)-dependent internalisation of GABA_ARs. Changes in surface GABA_ARs were measured by cell surface ELISA with anti-myc-specific antibody labelling the γ2 subunit, and presented as mean ± s.e.m. (n = 4). Statistical analysis was done using ANOVA with Bonferonni post-hoc test; *p < 0.05; n = number of independent experiments. l Schematic diagram of the GABA_AR/PLCδ/Ca²⁺/calcineurin feed-back mechanism underlying diazepam-dependent downregulation of GABA_ARs. According to this model, sustained activation of synaptic GABA_ARs by diazepam triggers a metabotropic, PLCδ/Ca²⁺/calcineurin signalling pathway which leads to receptor dephosphorylation by calcineurin, initiation of dynamin-dependent endocytosis resulting in a decrease in the size and number of postsynaptic GABA_AR clusters, and disassembly of inhibitory synapses. This mechanism is ‘switched off’ when PRIP1, PLCδ-related but catalytically inactive protein, outcompetes the PLCδ in binding to GABA_ARs, thereby preventing the activation of PLCδ and downstream Ca²⁺/calcineurin-dependent internalisation of these receptors