Fig. 2: AS283 inhibits CaMKII enzymatic function but does not impair GluN2B binding or synaptic localization.

Data are presented as mean values ± s.e.m. a, Schematic of the AS283 inhibitor mechanism of action: ATP-competitive inhibition blocks enzymatic activity while mimicking the nucleotide binding requirement to allow interaction with GluN2B. b, Representative immunoblot and quantification of in vitro kinase reactions at 30 °C in 1 mM ATP measuring purified CaMKIIα phosphorylation of GST–GluA1 S831 with varying concentrations of AS283 (0.41–10 μM; n = 4 independent concentration curves). For a negative control (Neg.), ATP was omitted. K_i values were derived from half-maximal inhibitory concentration (IC50) values of AS283-mediated inhibition of CaMKII phosphorylation of S831 and T286 using an experimentally derived K_m value of 33.3 μM (Extended Data Fig. 2b). c, Binding of purified CaMKIIα to immobilized GST–GluN2B-c was induced by Ca²⁺/CaM in the presence of 1 mM ADP alone or with 10 μM AS283 or 5 μM tatCN21 (n = 4 independent samples; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). ADP:AS283 P = 0.0697; ADP:tatCN21 P < 0.0001. ***P < 0.001. d, Confocal microscopy images of HEK293 cells co-expressing GFP–CaMKII and mCherry-2BC. Correlation indices before (Pre.) and after (Stim.) ionomycin-induced colocalization of GFP–CaMKII and mCherry-2BC under vehicle or 10 μM AS283 conditions (n = 18, 16 cells; repeated measures (RM) two-way ANOVA with Šídák’s multiple comparisons test). Scale bar, 10 μm. Vehicle P < 0.0001; AS283 P < 0.0001. ***P < 0.001. e, Confocal microscopy images of dissociated rat hippocampal cultures expressing GFP–CaMKII and mCh-PSD95 intrabody in the presence of 10 μM AS283 before and after cLTP (100 μM glutamate, 10 μM glycine; 45 s). CaMKII synaptic enrichment was measured before and after cLTP (n = 14,9 cells; RM two-way ANOVA with Šídák’s multiple comparisons test). Scale bar, 5 μm. Vehicle P < 0.0001; AS283 P < 0.0001. ***P  < 0.001.

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