CaMKII "autonomy" is required for initiating but not for maintaining neuronal long-term information storage

Abstract

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) "autonomy" (T286-autophosphorylation-induced Ca(2+)-independent activity) is required for long-term potentiation (LTP) and for learning and memory, as demonstrated by CaMKII T286A mutant mice. The >20-year-old hypothesis that CaMKII stimulation is required for LTP induction, while CaMKII autonomy is required for LTP maintenance was recently supported using the cell-penetrating fusion-peptide inhibitor antCN27. However, we demonstrate here that ant/penetratin fusion to CN27 compromised CaMKII-selectivity, by enhancing a previously unnoticed direct binding of CaM to ant/penetratin. In contrast to antCN27, the improved cell-penetrating inhibitor tatCN21 (5 mum) showed neither CaM binding nor inhibition of basal synaptic transmission. In vitro, tatCN21 inhibited stimulated and autonomous CaMKII activity with equal potency. In rat hippocampal slices, tatCN21 inhibited LTP induction, but not LTP maintenance. Correspondingly, tatCN21 also inhibited learning, but not memory storage or retrieval in a mouse in vivo model. Thus, CaMKII autonomy provides a short-term molecular memory that is important in the signal computation leading to memory formation, but is not required as long-term memory store.

Figure 1.

Ant/penetratin but not tat fusion compromised selectivity of CN inhibitor peptides by an additional CaM-directed mode of inhibition. A, The ant and tat sequences fused to the N terminus of CNs; (K) was replaced by the first K of CN27 in the fusion. B, Extent of CaMKII inhibition by 1 μm antCN27 and the reverse sequence control antRev depended on the CaM concentration, indicating an additional CaM-directed mode of inhibition generated by ant fusion. AC2 phosphorylation by CaMKII was measured. Error bars show SEM. C, tatCN21 (1 μm) strongly inhibited CaMKII-mediated AC3 phosphorylation at all CaM concentrations, while the reverse and scrambled sequence controls tatRev, and tatCtrl had no effect compared with assays without tat peptide. D, antCN27 blocked CaMKII activity, but also reduced CaMKIV activity by ∼70% when tested against a panel of different kinases.

Figure 2.

Fusion with a CN peptide enhanced the previously unrecognized direct CaM binding to ant/penetratin. A, Binding of biotinylated CaM (25 nm in TBS, pH 7.5, 1 mm CaCl₂) to proteins (CaMKII) and peptides (CBD, CN27, tat, and ant) immobilized by slot blot, in three different dilutions of the amount indicated. B, Interference of peptides (5 μm) with CaM binding (conditions as in A) to brain proteins in a blot overlay assay. The major Ca²⁺-dependent CaM-binding proteins were CaN-A and CaMKIIα. AntCN27 (top) and ant (bottom) affect Ca²⁺-dependent CaM binding, but not binding to a protein also detected after Ca²⁺ was chelated by EGTA. C, antCN27 and ant, but not tatCN21, compete with CBD for binding of TA-labeled CaM. Fluorescence of TA-CaM is reduced after addition of CBD (but not ant; supplemental Fig. 2, available at www.jneurosci.org as supplemental material). In presence of antCN27 or ant (but not tatCN21), significantly more CBD has to be added for the same reduction in fluorescence. Fluorescence (λ_ex = 335 nm; λ_em = 415 nm; 1 s sample time) was monitored for 150 s after each addition of CBD.

Figure 3.

tatCN21 inhibits stimulated and autonomous CaMKII activity with equally high potency and efficacy. A, tatCN21 potently inhibited Ca²⁺/CaM-stimulated CaMKII (2.5 nm) activity toward the peptide substrate syntide 2 (75 μm). The calculated IC₅₀ was ∼40 nm. B, tatCN21 inhibited stimulated and autonomous CaMKII activity with equal potency (as indicated by identical inhibition at 40 nm, the IC₅₀ calculated from A) and efficacy (as indicated by complete block of inhibition at 5 μm, 120-fold of IC₅₀), while stimulated activity of the related CaMKIV was not affected at all. Error bars indicate SEM in all panels.

Figure 4.

tatCN21 blocks induction but not maintenance of LTP. A, Perfusion of acute hippocampal slices (from 6- to 7-week-old rats) with tatCN21 (5 μm) did not affect basal transmission in CA1 but blocked LTP induction by high-HFS (2 × 1 s, 100 Hz) (n = 5), compared with untreated control (n = 5). fEPSP slope over time is shown as percentage of baseline. B, Paired pulse facilitation was the same before and after 20 min perfusion with tatCN21. Shown are sample traces from one experiment (left) and quantification of five experiments (right). C, Presynaptic fiber potential was unaffected by 20 min perfusion with tatCN21 (p > 0.2, paired t test). D, tatCN21 (5 μm) perfusion 15 min after LTP induction by HFS did not interfere with LTP maintenance. fEPSP slope is shown as percentage of baseline.

Figure 5.

tatCN21 interfered with learning but not memory in a hippocampus-dependent contextual fear conditioning paradigm. A, Intraperitoneal injection of tatCN21 (10 mg/kg) was done either 1 h before the training session (pre) or the test session 24 h after training (post). B, tatCN21 (n = 10) injection before the training session significantly reduced freezing behavior in the test session, compared with saline (n = 14) or the tatCtrl scrambled sequence control (n = 7) (**p < 0.05 in Bonferroni's multiple-comparisons test, after ANOVA with p < 0.023) (left). C, Injection of tatCN21 (n = 12) before the test session did not affect freezing behavior compared with injection of saline (n = 10) and tatCtrl control (n = 8) (p > 0.6, ANOVA) (right). Error bars indicate SEM in all panels.