Ca2+-dependent facilitation of Cav1.3 Ca2+ channels by densin and Ca2+/calmodulin-dependent protein kinase II.
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ABSTRACT:
SUBMITTER: Jenkins MA
PROVIDER: S-EPMC2990970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of synaptic responses in the postsynaptic density, but understanding of its mechanisms of action in the presynaptic neuron is incomplete. Here we show that CaMKII constitutively associates with and modulates voltage-gated calcium (Ca(V))2.1 channels that conduct P/Q type Ca(2+) currents and initiate transmitter release. Both exogenous and brain-specific inhibitors of CaMKII accelerate voltage-dependent inactivation, cause a negative shift in the voltage dependence of inactivation, and reduce Ca(2+)-dependent facilitation of Ca(V)2.1 channels. The modulatory effects of CaMKII are reduced by a peptide that prevents binding to Ca(V)2.1 channels but not by a peptide that blocks catalytic activity, suggesting that binding rather than phosphorylation is responsible for modulation. Our results reveal a signaling complex formed by Ca(V)2.1 channels and CaMKII that regulates P/Q-type Ca(2+) current in neurons. We propose an "effector checkpoint" model for the control of Ca(2+) channel fitness for function that depends on association with CaMKII, SNARE proteins, and other effectors of Ca(2+) signals. This regulatory mechanism would be important in presynaptic nerve terminals, where Ca(V)2.1 channels initiate synaptic transmission and CaMKII has noncatalytic effects on presynaptic plasticity.
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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.
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Project description:Calmodulin regulation of CaV channels is a prominent Ca(2+) feedback mechanism orchestrating vital adjustments of Ca(2+) entry. The long-held structural correlation of this regulation has been Ca(2+)-bound calmodulin, complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (individually transformed Langmuir analysis). Accordingly, we uncover frank exchange of Ca(2+)-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca(2+)-calmodulin binds an N-terminal spatial Ca(2+) transforming element module on the channel amino terminus, whereas the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.
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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.
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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.
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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.
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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.
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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.
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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.
| S-EPMC6725094 | biostudies-literature
Project description:The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase II? (CaMKII?) in cells. Although the C-terminal domain selectively binds CaMKII? in vitro, full-length densin associates with CaMKII? or CaMKII? in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKII?/? catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.
| S-EPMC3137056 | biostudies-literature