Fig. 4: The CaMKII mutation F89G impairs ATP binding but binds the ATP-competitive inhibitor NM-PP1 that restores GluN2B binding and LTP-induced synaptic translocation.
From: LTP induction by structural rather than enzymatic functions of CaMKII
Data are presented as mean values ± s.e.m. a, Schematic of the pharmacogenetic method of ATP-competitive CaMKII inhibition; enlargement of the CaMKII ATP-binding pocket by the F89G mutation allows for selective binding of the ATP-competitive inhibitor NM-PP1. b, Immunoblots of in vitro kinase reactions at 30 °C including GFP–CaMKII WT, K42M and F89G with GST–GluA1-c tail. Reactions were performed either with vehicle control or with 10 μM NM-PP1 (n = 1) (related independent experiments are in Extended Data Fig. 4a). c, Confocal microscopy images of HEK293 cells co-expressing GFP–CaMKII F89G and mCherry-2BC in the presence of vehicle or 10 μM NM-PP1 before and after ionomycin stimulation. Correlation indices were measured before and after Ionomycin-induced colocalization of GFP–CaMKII (WT, K42M and F89G) with mCherry-2BC, represented as change in the correlation index (vehicle: n = 22, 17, 12 cells; NM-PP1: n = 20, 24, 13 cells; one-way ANOVA, Tukey’s multiple comparisons test). Scale bar, 10 μm. For vehicle, WT:F89G P < 0.0001, WT:K42M P < 0.0001 and F89G:K42M P = 0.7333. For NM-PP1, WT:F89G P = 0.7771, WT:K42M P = 0.0009 and F89G:K42M P < 0.0001. ***P < 0.001. d, Confocal microscopy images of dissociated rat hippocampal cultures expressing GFP–CaMKII (WT, K42M or F89G) and mCh-PSD95 intrabody before and after cLTP. CaMKII synaptic enrichment was measured before and after cLTP (n = 13, 14, 14 cells; RM two-way ANOVA with Šídák’s multiple comparisons test). Scale bar, 5 μm. For basal, WT:K42M P = 0.0022, WT:F89G P = 0.0655 and K42M:F89G P = 0.5478. For cLTP, WT:K42M P < 0.0001, WT:F89G P = 0.0004 and K42M:F89G P = 0.9562. **P < 0.01, ***P < 0.001. e, Confocal microscopy images before and after cLTP as in panel d but in the presence of 10 μM NM-PP1. CaMKII synaptic enrichment was measured before and after cLTP (n = 7, 6, 7 cells; RM two-way ANOVA with Šídák’s multiple comparisons test). Scale bar, 5 μm. For basal, WT:K42M P = 0.0079, WT:F89G P = 0.6909 and K42M:F89G P = 0.0009. For cLTP, WT:K42M P < 0.0001, WT:F89G P = 0.9582 and K42M:F89G P < 0.0001. **P < 0.01, ***P < 0.001.
