Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II.
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ABSTRACT:
SUBMITTER: Scott-Woo GC
PROVIDER: S-EPMC1131441 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description:Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camuialpha detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camuialpha can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.
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Project description:Ca(v)1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca(2+) signaling in neurons. CaMKII directly potentiates the activity of Ca(v)1.2 and Ca(v)1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca(2+)-dependent facilitation of Ca(v)1.3 channels. While neither CaMKII nor densin independently affects Ca(v)1.3 properties in transfected HEK293T cells, the two together augment Ca(v)1.3 Ca(2+) currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca(2+), CaMKII activation, and its association with densin, as well as densin binding to the Ca(v)1.3 alpha(1) subunit C-terminal domain. Ca(v)1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca(2+)-dependent facilitation that may intensify postsynaptic Ca(2+) signals during high-frequency stimulation.
| S-EPMC2990970 | biostudies-literature
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+ sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+ signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287 (T286? isoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287 autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287 autophosphorylation. Our data suggest T287 autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.
| S-EPMC5748111 | biostudies-literature
Project description:AimsCalmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood.Methods and resultsPurified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function.ConclusionCa2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.
| S-EPMC2802203 | biostudies-literature
Project description: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 bindup 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.
| S-EPMC2820514 | biostudies-literature
Project description:Calmodulin sits at the center of molecular mechanisms underlying learning and memory. Its complex and sometimes opposite influences, mediated via the binding to various proteins, are yet to be fully understood. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) both bind open calmodulin, favoring Long-Term Potentiation (LTP) or Depression (LTD) respectively. Neurogranin binds to the closed conformation of calmodulin and its impact on synaptic plasticity is less clear. We set up a mechanistic computational model based on allosteric principles to simulate calmodulin state transitions and its interactions with calcium ions and the three binding partners mentioned above. We simulated calcium spikes at various frequencies and show that neurogranin regulates synaptic plasticity along three modalities. At low spike frequencies, neurogranin inhibits the onset of LTD by limiting CaN activation. At intermediate frequencies, neurogranin facilitates LTD, but limits LTP by precluding binding of CaMKII with calmodulin. Finally, at high spike frequencies, neurogranin promotes LTP by enhancing CaMKII autophosphorylation. While neurogranin might act as a calmodulin buffer, it does not significantly preclude the calmodulin opening by calcium. On the contrary, neurogranin synchronizes the opening of calmodulin's two lobes and promotes their activation at specific frequencies. Neurogranin suppresses basal CaN activity, thus increasing the chance of CaMKII trans-autophosphorylation at high-frequency calcium spikes. Taken together, our study reveals dynamic regulatory roles played by neurogranin on synaptic plasticity, which provide mechanistic explanations for opposing experimental findings.
| S-EPMC7041932 | biostudies-literature
Project description:Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the "closed" Ca2+-free conformation. We have measured their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation. We show that CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways.
| S-EPMC1599897 | biostudies-literature
Project description:Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current (I(f)) and by promoting inward Na(+)/Ca(2+) exchanger current (I(NCX)) by a "Ca(2+) clock" mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect I(f) and Ca(2+) clock mechanisms are uncertain and controversial, but the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to ?-adrenergic receptor (?-AR) stimulation. The aim of this study was to measure the contribution of the Ca(2+) clock and CaMKII to cardiac pacing independent of ?-AR agonist stimulation.We used the L-type Ca(2+) channel agonist Bay K8644 (BayK) to activate the SANC Ca(2+) clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca(2+) current (I(Ca)) equally in all SANCs, indicating that increasing I(Ca) was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase I(f) or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca(2+) by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca(2+) release and I(NCX) were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups.The Ca(2+) clock is capable of supporting physiological fight-or-flight responses, independent of ?-AR stimulation or I(f) increases. Complete Ca(2+) clock and ?-AR stimulation responses require CaMKII.
| S-EPMC3116039 | biostudies-literature
Project description:The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.
| S-EPMC3102786 | biostudies-literature
Project description:Ca(2+)/calmodulin dependent protein kinase II (CaMKII) is a broadly distributed metazoan Ser/Thr protein kinase that is important in neuronal and cardiac signaling. CaMKII forms oligomeric assemblies, typically dodecameric, in which the calcium-responsive kinase domains are organized around a central hub. We review the results of crystallographic analyses of CaMKII, including the recently determined structure of a full-length and autoinhibited form of the holoenzyme. These structures, when combined with other data, allow informed speculation about how CaMKII escapes calcium-dependence when calcium spikes exceed threshold frequencies.
| S-EPMC3788167 | biostudies-literature