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. 2012 Dec 5;32(49):17874–17881. doi: 10.1523/JNEUROSCI.2535-12.2012

Figure 4.

Figure 4.

Characterization of δ intracellular domain. A, Putative μ2 binding domains within the GABAA-R δ subunit ICD. The entire rat δ ICD (aa 316–410) is shown. A classical YxxΦ motif (375YRSV) is located at residues 375–378, while an atypical basic rich motif similar to those seen in GABAA-R β(1–3) is found at residues 322–334. Both are highlighted. B, Diagram of GST δ ICD constructs in this study: 1, GST only; 2, GST+ δICD full-length residues 316–410 (δ); 3, GST + first half of δ ICD residues 316–359 (δR); 4, GST + second half δ ICD residues 360–410 (δY); 5, GST + δ full-length R/K piece mutated residues 322–326 (δ322RKKRK→GAAGA); 6, GST + δ full-length Y375 mutated to A (δ375Y→A); 7, GST + δ full-length, where both the 322R/K piece and 375Y are mutated (δ2X mutant). C, Each μ2 binding domain of the δ ICD binds to μ2. GST-only (control) does not bind to μ2, but constructs 2–6 all bind to μ2. This includes all δ ICD constructs containing at least one complete μ2-binding domain (322R/K region only shown as δR; 375YRSV-only shown as δY). Only the construct lacking both μ2-binding domains shows no binding to μ2. Therefore, each of the two μ2 binding domains directly bind to μ2. Quantification of binding shows there are no significant differences between mutants and δ ICD full-length, except for the double mutant, which significantly lost all μ2 binding (*p < 0.001; one-way ANOVA). All other constructs are significantly different from the double mutant (p < 0.05; one-way ANOVA). D, The 375YRSV-motif of the δ ICD detects lower [μ2]. The 1 nm, 0.1 μm, and 0.1 mm AP2 fractions were incubated with the GST-δ truncation proteins to see if one region binds at a lower μ2 concentration than the other. The procedure and analysis were similar to those done with the pull-down experiments, with representative blot and cumulative quantified μ2 binding plot shown here. The 1 nm concentration showed no binding, but 0.1 μm showed μ2 binding more to the construct lacking the basic rich region, suggesting this lower-concentration binding is more influenced by the 375YRSV-motif. The difference between binding seemed to be undetectable at higher μ2 concentrations, likely because the basic-rich 322R/K motif is now participating in its own μ2 binding (p < 0.05; one-way ANOVA; N = 3).