Fig. 3: Both γ8 PDZ and GluA2 NTD interactions are sufficient for AMPAR synaptic transmission.
From: AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions
a AMPAR knockout and rescue strategy in Gria1-3fl/fl tissue. AMPARs in in vivo AAV-EGFP-Cre-transduced organotypic hippocampal slices (left, outline depicted dashed) are rescued by SCE (right) with coexpressed tdTomato for cell identification. Dual recordings are performed between unmodified (Untransduced and untransfected—Untrans.) and rescued (EGFP & tdTomato positive) neurons. b Cre-transduction abolishes AMPAR synaptic transmission (Untrans.: 1.00 ± 0.20, Cre: 0.07 ± 0.03, n = 7 pairs, p = 0.016). GluA1_γ8 transfection rescues synaptic transmission (Untrans.: 1.00 ± 0.29, GluA1_γ8 rescue: 0.72 ± 0.20, n = 8 pairs, p = 0.25); however, γ8 PDZ mutation prevents this rescue (Untrans.: 1.00 ± 0.20, GluA1_γ8 ΔPDZ rescue: 0.09 ± 0.02, n = 9 pairs, p = 0.0039). Example traces are depicted with stimulation indicated (triangle). c Rescue of AMPAR-null cells by GluA2Q_γ8 produces robust synaptic transmission (Untrans.: −57.2 ± 15.0 pA, GluA2Q_γ8 rescue: −40.9 ± 6.4 pA, n = 10 pairs, p = 0.77). d γ8 PDZ deletion reduces but does not prevent AMPAR transmission (Untrans.: −65.9 ± 13.1 pA, GluA2Q_γ8 ΔPDZ rescue: −22.9 ± 4.0 pA, n = 8 pairs, p = 0.0078). e Removal of both the γ8 PDZ ligand and GluA2 NTD prevents rescue of AMPAR synaptic transmission (Untrans.: −84.4 ± 24.5 pA, GluA2Q_γ8 ΔPDZ ΔNTD rescue: −4.5 ± 1.4 pA, n = 5 pairs, p = 0.63). f Robust transmission is observed on rescue of AMPAR knockout by heteromeric GluA1_γ8/GluA2_γ8 receptors (Untrans.: −80.0 ± 9.6 pA, A1_γ8/A2_γ8 rescue: −65.2 ± 10.9 pA, n = 11 pairs, p = 0.10). g γ8 PDZ deletion from both GluA1 and GluA2 rescue constructs reduces but does not prevent AMPAR transmission (Untrans.: -102.7 ± 8.7 pA, A1_γ8/A2_γ8 ΔPDZs rescue: −28.4 ± 3.6 pA, n = 14 pairs, p = 0.0001). h Exchanging the GluA2 NTD for that of GluA1 to remove GluA2 NTD from heteromeric AMPAR rescue prevents any rescue of transmission (Untrans.: −77.4 ± 8.5 pA, A1_γ8/A2A1NTD_γ8 ΔPDZs rescue: −5.8 ± 1.5 pA, n = 6 pairs, p = 0.031). Normalised GluA2Q_γ8 (i) and heteromeric (j) rescue experiments (I Untrans.: 1.00 ± 0.26, GluA2Q_γ8 rescue: 0.72 ± 0.11; Untrans.: 1.00 ± 0.20, GluA2Q_γ8 ΔPDZ rescue: 0.35 ± 0.06; Untrans.: 1.00 ± 0.29, GluA2Q_γ8 ΔPDZ ΔNTD rescue: 0.05 ± 0.02. j Untrans.: 1.00 ± 0.12, A1_γ8/A2_γ8 rescue: 0.81 ± 0.14; Untrans.: 1.00 ± 0.08, A1_γ8/A2_γ8 ΔPDZs rescue: 0.28 ± 0.04; Untrans.: 1.00 ± 0.11, A1_γ8/A2A1NTD_γ8 ΔPDZs rescue: 0.07 ± 0.02). Transmission levels in Cre only expressing cells are depicted as a line. k The GluA2 NTD drives synaptic anchoring of GluA1_γ8 ΔPDZ receptors (Amplitudes—Untrans.: −80.7 ± 12.8 pA, GluA1A2NTD_γ8 ΔPDZ: −47.1 ± 5.8 pA, n = 11 pairs, p = 0.042. Normalised data—Untrans.: 1.00 ± 0.16, GluA1A2NTD_γ8 ΔPDZ: 0.58 ± 0.07). * indicates p < 0.05, **p < 0.01, ***p < 0.001 and ns specifies no significance. All data are presented as mean ± SEM, and all statistics are performed with a Wilcoxon matched-pairs signed-rank test. Source data are provided as a source data file.
