Conformational changes underlying calcium/calmodulin-dependent protein kinase II activation

Calcium/calmodulin-dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.     

Dissecting cooperative calmodulin binding to CaM kinase II: a detailed stochastic model

Calmodulin (CaM) is a major Ca²⁺ binding protein involved in two opposing processes of synaptic plasticity of CA1 pyramidal neurons: long-term potentiation (LTP) and depression (LTD). The N- and C-terminal lobes of CaM bind to its target separately but cooperatively and introduce complex dynamics that cannot be well understood by experimental measurement. Using a detailed stochastic model constructed upon experimental data, we have studied the interaction between CaM and Ca²⁺-CaM-dependent protein kinase II (CaMKII), a key enzyme underlying LTP. The model suggests that the accelerated binding of one lobe of CaM to CaMKII, when the opposing lobe is already bound to CaMKII, is a critical determinant of the cooperative interaction between Ca²⁺, CaM, and CaMKII. The model indicates that the target-bound Ca²⁺ free N-lobe has an extended lifetime and may regulate the Ca²⁺ response of CaMKII during LTP induction. The model also reveals multiple kinetic pathways which have not been previously predicted for CaM-dissociation from CaMKII.     

STDP in a bistable synapse model based on CaMKII and associated signaling pathways

The calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in the induction of long-term postsynaptic modifications following calcium entry. Experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states. The biochemical network involving CaMKII and its regulating protein signaling cascade has been hypothesized to durably maintain the evoked synaptic state in the form of a bistable switch. However, it is still unclear whether experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such a network. We present a detailed biochemical model of the CaMKII autophosphorylation and the protein signaling cascade governing the CaMKII dephosphorylation. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentration, and high calcium transients can switch the system from the weakly phosphorylated (DOWN) to the highly phosphorylated (UP) state of the CaMKII (similar to a LTP event). We show here that increased CaMKII dephosphorylation activity at intermediate Ca²⁺ concentrations can lead to switching from the UP to the DOWN state (similar to a LTD event). This can be achieved if protein phosphatase activity promoting CaMKII dephosphorylation activates at lower Ca²⁺ levels than kinase activity. Finally, it is shown that the CaMKII system can qualitatively reproduce results of plasticity outcomes in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. This shows that the CaMKII protein network can account for both induction, through LTP/LTD-like transitions, and storage, due to its bistability, of synaptic changes.     

Mechanistic Investigation of the Arrhythmogenic Role of Oxidized CaMKII in the Heart

Oxidative stress and calcium (Ca²⁺)/calmodulin (CaM)-dependent protein kinase II (CaMKII) both play important roles in the pathogenesis of cardiac disease. Although the pathophysiological relevance of reactive oxygen species (ROS) and CaMKII has been appreciated for some time, recent work has shown that ROS can directly oxidize CaMKII, leading to its persistent activity and an increase of the likelihood of cellular arrhythmias such as early afterdepolarizations (EADs). Because CaMKII modulates the function of many proteins involved in excitation-contraction coupling, elucidation of its role in cardiac function, in both healthy and oxidative stress conditions, is challenging. To investigate this role, we have developed a model of CaMKII activation that includes both the phosphorylation-dependent and the newly identified oxidation-dependent activation pathways. This model is incorporated into our previous local-control model of the cardiac myocyte that describes excitation-contraction coupling via stochastic simulation of individual Ca²⁺ release units and CaMKII-mediated phosphorylation of L-type Ca²⁺ channels (LCCs), ryanodine receptors and sodium (Na⁺) channels. The model predicts the experimentally measured slow-rate dependence of H₂O₂-induced EADs. Upon increased H₂O₂, simulations suggest that selective activation of late Na⁺ current (I_NaL), although it prolongs action potential duration, is not by itself sufficient to produce EADs. Similar results are obtained if CaMKII effects on LCCs and ryanodine receptors are considered separately. However, EADs emerge upon simultaneous activation of both LCCs and Na⁺ channels. Further modeling results implicate activation of the Na⁺-Ca²⁺ exchanger (NCX) as an important player in the generation of EADs. During bradycardia, the emergence of H₂O₂-induced EADs was correlated with a shift in the timing of NCX current reversal toward the plateau phase earlier in the action potential. Using the timing of NCX current reversal as an indicator event for EADs, the model identified counterintuitive ionic changes—difficult to experimentally dissect—that have the greatest influence on ROS-related arrhythmia propensity.     

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

Because CaMKII is the critical Ca²⁺ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca²⁺ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.     

CaM kinase II as frequency decoder of Ca2+ oscillations

In many cell types, Ca²⁺ signals are organized in the form of repetitive spikes. The frequency of these intracellular Ca²⁺ oscillations increases with the level of stimulation, suggesting the existence of a frequency encoding phenomenon. The question arises as to how the frequency of Ca²⁺ oscillations can be decoded inside the cell. Ca²⁺/calmodulin kinase II has long been proposed as an attractive candidate, as it is a key target of Ca²⁺ signals. By immobilizing the Ca²⁺/calmodulin kinase II and subjecting it to pulses of Ca²⁺ of variable amplitude, duration, and frequency, De Koninck and Schulman⁽¹⁾ have shown for the first time that the autonomous activity of Ca²⁺/calmodulin kinase II is highly sensitive to the temporal pattern of Ca²⁺ oscillations. BioEssays 20 :607–610, 1998.© 1998 John Wiley & Sons Inc.     

Participation of CaMKII in neuronal plasticity and memory formation

1. The unique biochemical properties of Ca²⁺/calmodulin (CaM)-dependent protein kinase II have made this enzyme one of the paradigmatic models of the forever searched memory molecule.2. In particular, the central participation of CaMKII as a sensor of the Ca²⁺ signals generated by activation of NMDA receptors after the induction of long-term plastic changes, has encouraged the use of pharmacological, genetic, biochemical, and imaging tools to unveil the role of this kinase in the acquisition, consolidation, and expression of different types of memories.3. Here we review some of the more exciting discoveries related to the mechanisms involved in CaMKII activation and synaptic plasticity.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Synergy between CaMKII substrates and β-adrenergic signaling in regulation of cardiac myocyte Ca(2+) handling

Cardiac excitation-contraction coupling is a highly coordinated process that is controlled by protein kinase signaling pathways, including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA). Increased CaMKII expression and activity (as occurs during heart failure) destabilizes EC coupling and may lead to sudden cardiac death. To better understand mechanisms of cardiac CaMKII function, we integrated dynamic CaMKII-dependent regulation of key Ca²⁺ handling targets with previously validated models of cardiac EC coupling, Ca²⁺/calmodulin-dependent activation of CaMKII, and β-adrenergic activation of PKA. Model predictions are validated against CaMKII-overexpression data from rabbit ventricular myocytes. The model demonstrates how overall changes to Ca²⁺ handling during CaMKII overexpression are explained by interactions between individual CaMKII substrates. CaMKII and PKA activities during β-adrenergic stimulation may synergistically facilitate inotropic responses and contribute to a CaMKII-Ca²⁺-CaMKII feedback loop. CaMKII regulated early frequency-dependent acceleration of relaxation and EC coupling gain (which was highly sarcoplasmic reticulum Ca²⁺ load-dependent). Additionally, the model identifies CaMKII-dependent ryanodine receptor hyperphosphorylation as a proarrhythmogenic trigger. In summary, we developed a detailed computational model of CaMKII and PKA signaling in cardiac myocytes that provides unique insights into their regulation of normal and pathological Ca²⁺ handling.     

Lobe-specific functions of Ca2+·calmodulin in alphaCa2+·calmodulin-dependent protein kinase II activation

N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.     

A simulation study on the activation of cardiac CaMKII delta-isoform and its regulation by phosphatases

Although the highly conserved Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is known to play an essential role in cardiac myocytes, its involvement in the frequency-dependent acceleration of relaxation is still controversial. To investigate the functional significance of CaMKII autophosphorylation and its regulation by protein phosphatases (PPs) in heart, we developed a new mathematical model for the CaMKIIδ isoform. Due to better availability of experimental data, the model was first adjusted to the kinetics of the neuronal CaMKIIα isoform and then converted to a CaMKIIδ model by fitting to kinetic data of the δ isoform. Both models satisfactorily reproduced experimental data of the CaMKII-calmodulin interaction, the autophosphorylation rate, and the frequency dependence of activation. The level of autophosphorylated CaMKII cumulatively increased upon starting the Ca²⁺ stimulation at 3 Hz in the δ model. Variations in PP concentration remarkably affected the frequency-dependent activation of CaMKIIδ, suggesting that cellular PP activity plays a key role in adjusting CaMKII activation in heart. The inhibitory effect of PP was stronger for CaMKIIα compared to CaMKIIδ. Simulation results revealed a potential involvement of CaMKIIδ autophosphorylation in the frequency-dependent acceleration of relaxation at physiological heart rates and PP concentrations.     

In Vitro Reconstitution of a CaMKII Memory Switch by an NMDA Receptor-Derived Peptide

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca²⁺/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca²⁺ and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca²⁺ range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.     

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca²⁺-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.     

On the Mechanism of Synaptic Depression Induced by CaMKIIN,an Endogenous Inhibitor of CaMKII

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca²⁺ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.     

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Autonomous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca²⁺ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca²⁺ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca²⁺ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations.MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca²⁺ leak (in the form of spontaneous Ca²⁺ waves) under basal conditions and that Ca²⁺ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [³H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca²⁺ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca²⁺ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.     

In?Vitro Reconstitution of a CaMKII Memory Switch by an NMDA Receptor-Derived Peptide

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca²⁺/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca²⁺ and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca²⁺ range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.     

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C‐terminal regions of second intracellular loops. This binding relies on Ca²⁺ activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca²⁺ recruits CaMKIIα to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity‐dependent manner. Our data identify a new model of protein–protein interactions. In a Ca²⁺‐sensitive manner, CaMKIIα regulates M4R efficacy and controls the acetylcholine–dopamine balance in the basal ganglia and also the dynamics of movement.     

A Dynamic Model of Interactions of Ca2+, Calmodulin,and Catalytic Subunits of Ca2+/Calmodulin-Dependent Protein Kinase II

During the acquisition of memories, influx of Ca²⁺ into the postsynaptic spine through the pores of activated N-methyl-d-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca²⁺ influx during the first few seconds of activity is interpreted within the Ca²⁺-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity, including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bind up to 4 Ca²⁺ ions. As a first step toward clarifying how the Ca²⁺-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca²⁺, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca²⁺ play a significant role in activation of CaMKII in the physiological regime, supporting the notion that processing of Ca²⁺ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca²⁺ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca²⁺ transients arises from the kinetics of interaction of fluctuating Ca²⁺ with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.     

Translocation of CaMKII to dendritic microtubules supports the plasticity of local synapses

The processing of excitatory synaptic inputs involves compartmentalized dendritic Ca²⁺ oscillations. The downstream signaling evoked by these local Ca²⁺ transients and their impact on local synaptic development and remodeling are unknown. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an important decoder of Ca²⁺ signals and mediator of synaptic plasticity. In addition to its known accumulation at spines, we observed with live imaging the dynamic recruitment of CaMKII to dendritic subdomains adjacent to activated synapses in cultured hippocampal neurons. This localized and transient enrichment of CaMKII to dendritic sites coincided spatially and temporally with dendritic Ca²⁺ transients. We show that it involved an interaction with microtubular elements, required activation of the kinase, and led to localized dendritic CaMKII autophosphorylation. This process was accompanied by the adjacent remodeling of spines and synaptic AMPA receptor insertion. Replacement of endogenous CaMKII with a mutant that cannot translocate within dendrites lessened this activity-dependent synaptic plasticity. Thus, CaMKII could decode compartmental dendritic Ca²⁺ transients to support remodeling of local synapses.     

Regulation of phosphorylation at the postsynaptic density during different activity states of Ca/calmodulin-dependent protein kinase II

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca²⁺ and, once autophosphorylated on T-286/7, remains active in the absence of Ca²⁺ (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca²⁺, and (3) active in the absence of Ca²⁺. The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca²⁺ varied. Most notably, autonomous activity in the absence of Ca²⁺ was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.