Calmodulin Activity Research Articles

Visualizing CaMKII and CaM activity: a paradigm of compartmentalized signaling

Calcium (Ca(2+)) has long been recognized as a crucial intracellular messenger attaining stimuli-specific cellular outcomes via localized signaling. Ca(2+)-binding proteins, such as calmodulin (CaM), and its target proteins are key to the segregation and refinement of these Ca(2+)-dependent signaling events. This review not only summarizes the recent technological advances enabling the study of subcellular Ca(2+)-CaM and Ca(2+)-CaM-dependent protein kinase (CaMKII) signaling events but also highlights the outstanding challenges in the field.

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.

Nonthermal electromagnetic fields: From first messenger to therapeutic applications

Nonthermal pulsed electromagnetic fields, from low frequency to pulse-modulated radio frequency, have been successfully employed as adjunctive therapy for the treatment of delayed and non-union fractures, fresh fractures and chronic wounds. Recent increased understanding of the mechanism of action of electromagnetic fields (EMF) has permitted technologic advances allowing the development of EMF devices which are portable and disposable, can be incorporated into dressings, supports and casts, and can be used over clothing. This broadens the use of non-pharmacological, non-invasive EMF therapy to the treatment of postoperative pain and edema to enhance surgical recovery. EMF therapy is rapidly becoming a standard part of surgical care, and new, more significant, clinical applications for osteoarthritis, brain and cardiac ischemia and traumatic brain injury are in the pipeline. This study reviews recent evidence which suggests that calmodulin (CaM)-dependent nitric oxide signaling is involved in cell and tissue response to weak nonthermal EMF signals. There is abundant evidence that EMF signals can be configured a priori to increase the rate of CaM activation, which, in turn, can modulate the biochemical cascades living cells and tissues employ in response to external insult. Successful applications in pilot clinical trials, coupled with evidence at the cellular and animal levels, provide support that EMF is a first messenger that can modulate the response of challenged biological systems.

Calcium signaling mediates cold sensing in insect tissues.

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

Calmodulin activation of polo-like kinase 1 is required during mitotic entry

Polo-like kinase 1 (Plk1) is a conserved key regulator of the G2/M transition, but its upstream spatiotemporal regulators remain unknown. With the help of immunofluorescence, co-immunoprecipitation, and glutathione S-transferase (GST) pull-down assay, we found that calmodulin (CaM) is one such regulatory molecule that associates with Plk1 from G2 to metaphase. More importantly, this interaction results in considerable stimulation of Plk1 kinase activity leading to hyperphosphorylation of Cdc25C. Our results provide new insight into the role of CaM as an upstream regulator of Plk1 activation during mitotic entry.

Suramin Distinguishes Between the Calmodulin Binding Sequences of Inducible and Neuronal Nitric Oxide Synthase

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide (•NO) from L‐arginine in several tissues in the body. Nitric oxide is believed to play a critical role in cardiovascular disease, stroke, and cancer. For this reason, selective inhibition of NOS isoforms is currently under investigation for their potential as targets in drug design. All known isoforms of NOS require binding of calmodulin (CaM), a calcium binding protein ubiquitously expressed in eukaryotes, for activity. The interactions among the NOS isoforms and CaM vary, and therefore pose a good place to study in differentiating these enzymes. We have previously demonstrated that suramin, a polysulfonated naphthylurea, can distinguish between the CaM binding region of the inducible NOS and the neuronal NOS. When bound to NOS, suramin inhibits its activity by preventing CaM binding and activation. Suramin's inhibition was found not to be time dependent, and IC50 values were found to be ~8 μM and ~120 μM for the inducible NOS and neuronal NOS, respectively. SDS‐PAGE and Western analyses demonstrated that suramin is able to inhibit the interaction between NOS and CaM. AutoDoc analysis further demonstrated that suramin most likely interacts with a portion of the NOS molecule believed to be crucial to CaM's interaction. These studies implicate suramin as a potential lead compound.

Spatiotemporal Dynamics of Calmodulin in Dendritic Spines during Calcium Influx

Experiments by Lee et al. (Lee et al., 2009) provide compelling evidence of spatially inhomogeneous reactant distributions in dendritic spines. We have built a reaction-diffusion model of a dendritic spine using the calmodulin activation kinetics developed by Faas et al. (Faas et al., 2011). Upon calcium influx from voltage-sensitive calcium channels, we find spatial inhomogeneities lasting 2 ms in the distribution of calmodulin with N-lobe-bound calcium, but no significant inhomogeneities in the distributions of calmodulin with C-lobe-bound calcium or calbindin. In the presence of 5 mM EGTA, the spatial inhomogeneity in the concentration of calmodulin with N-lobe-bound calcium persists for 1 ms, eventually becoming restricted to the outer 200 nm of the spine at 20 mM EGTA. Calmodulin with C-lobe-bound calcium continues to penetrate to the interior, but at a lower concentration and with an inhomogeneous distribution. In the presence of 5 mM and 20 mM BAPTA, concentrations of both species drop sharply and they are not able to diffuse to the interior of the spine. We believe that these spatial inhomogeneities (arising during calcium influx) can assist in explaining the results presented by Lee et al. Furthermore, our work forms a basis for a spatially-resolved reaction-diffusion model of dendritic spines that includes the reactions underlying the activation, phosphorylation and dephosphorylation of CaMKII, as well as long-term potentiation, and long-term depression.1. S-J. R. Lee et al., Activation of CaMKII in single dendritic spines during long-term potentiation. NATURE, 458(7236):299-U58, MAR 19 2009.2. G. C. Faas., Calmodulin as a direct detector of Ca2+ signals. NATURE Neuroscience, 14(3):301-304, MAR 2011.

ANO1 Activation does not require Calmodulin

The Ca2+-activated Cl channel anoctamin-1 (ANO1, also called TMEM16A), a member of the Anoctamin superfamily, plays a variety of important physiological roles including epithelial fluid secretion. ANO1 is activated and gated by intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to ANO1 or whether an additional Ca2+-binding subunit such as calmodulin (CaM) is required. Here, we report that CaM is not a necessary component for ANO1 activation for the following reasons: 1) ANO1 activation by photolysis of caged Ca2+ is very rapid (<1ms), suggesting that Ca2+ binds directly to the channel. 2) Exogenous CaM has no effect on the currents generated by either the ac or abc isoforms of ANO1 in inside-out excised patches. 3) Over-expression of functionally defective CaM has no effect on ANO1 (abc isoform) currents while it eliminates the small conductance Ca-activated K (SK2) current. 4) CaM does not physically interact with ANO1 as determined by co-immunoprecipitation. 5) ANO1 is activated in excised patches by Ba, which is a very poor CaM activator. We also re-examined the effect of the CaM inhibitor trifluoperazine (TFP). TFP blocks ANO1 in a voltage-dependent manner, suggesting that TFP blocks the current by lodging in the ANO1 pore. These results show CaM is not required for ANO1 activation and supports the hypothesis that Ca binds directly to ANO1.

5-Fluorouracil signaling through a calcium–calmodulin-dependent pathway is required for p53 activation and apoptosis in colon carcinoma cells

5-Fluorouracil (5-FU) is an anti-metabolite that is in clinical use for treatment of several cancers. In cells, it is converted into three distinct fluoro-based nucleotide analogs, which interfere with DNA synthesis and repair, leading to genome impairment and, eventually, apoptotic cell death. Current knowledge states that in certain cell types, 5-FU-induced stress is signaling through a p53-dependent induction of tumor necrosis factor-receptor oligomerization required for death-inducing signaling complex formation and caspase-8 activation. Here we establish a role of calcium (Ca(2+)) as a messenger for p53 activation in response to 5-FU. Using a combination of pharmacological and genetic approaches, we show that treatment of colon carcinoma cells stimulates entry of extracellular Ca(2+) through long lasting-type plasma membrane channels, which further directs posttranslational phosphorylation of at least three p53 serine residues (S15, S33 and S37) by means of calmodulin (CaM) activity. Obstructing this pathway by the Ca(2+)-chelator BAPTA (1,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid) or by inhibitors of CaM efficiently reduces 5-FU-induced caspase activities and subsequent cell death. Moreover, ectopic expression of p53 S15A in HCT116 p53(-/-) cells confirmed the importance of a Ca(2+)-CaM-p53 axis in 5-FU-induced extrinsic apoptosis. The fact that a widely used therapeutic drug, such as 5-FU, is operating via this pathway could provide new therapeutic intervention points, or specify new combinatorial treatment regimes.

JAK2 mediates the acute response to decreased cell volume in mouse preimplantation embryos by activating NHE1

Preimplantation mouse embryos are particularly sensitive to increased osmolarity within their normal physiological range. The detrimental effects can be alleviated by organic osmolytes such as glycine transported into early embryos, an effect thought to be due to the organic osmolyte replacing a portion of intracellular inorganic ions accumulated during acute cell volume regulation. However, no mechanism of cell volume regulation dependent on inorganic ions has been identified in preimplantation embryos. We found that decreased cell volume rapidly activated Na(+)/H(+) exchange in preimplantation mouse embryos. This activity was likely mediated by the NHE1 (Slc9a1) isoform, since it was blocked by the highly selective NHE1 inhibitor, cariporide, which also inhibited the ability of the 1-cell embryo to maintain cell volume. How NHE1 is activated by decreased cell volume is not generally well understood. Full activation of NHE1 by decreased cell volume in 2-cell mouse embryos required the activity of the tyrosine kinase Janus kinase 2 (Jak2), since NHE1 activation was inhibited by the general tyrosine kinase inhibitor genistein, several selective inhibitors of Jak2, and dominant negative Jak2 expressed in 2-cell embryos. Decreased cell volume furthermore resulted in increased tyrosine phosphorylation of proteins in 2-cell embryos detected both by anti-phosphotyrosine antibody and an antibody directed against active phospho-Jak2. Thus, Jak2 apparently serves as a cell volume sensor in embryos. Evidence from pharmacological inhibitors further indicated that NHE1 activation by decreased cell volume was dependent on calmodulin activity, likely downstream of Jak2, and required active phospholipase C.

Modelling Ca2+ -dependent proteins in the spine - challenges and solutions

Background / Purpose: Modelling post-synaptic proteins poses three technical problems: small absolute molecule numbers, large numbers of possible states, and the complex geometry of the spine, which is not a well-mixed compartment. Computational approaches are needed that solve all three of these problems. Main conclusion: Stochastic simulation methods can be used for systems with small molecule numbers, agent-based methods to represent multi-state molecules, and spatial methods to simulate events in complex geometries. We used the agent-based spatial stochastic simulator MCell to model the Ca2+-dependent activation of calmodulin and Ca2+/calmodulin-dependent kinase II (CaMKII) in the spine. Next steps: Next steps will include the extension of our model to include more interaction partners, and to represent some of the regulation events in more detail.

Delay of Iris flower senescence by cytokinins and jasmonates

It is not known whether tepal senescence in Iris flowers is regulated by hormones. We applied hormones and hormone inhibitors to cut flowers and isolated tepals of Iris × hollandica cv. Blue Magic. Treatments with ethylene or ethylene antagonists indicated lack of ethylene involvement. Auxins or auxin inhibitors also did not change the time to senescence. Abscisic acid (ABA) hastened senescence, but an inhibitor of ABA synthesis (norflurazon) had no effect. Gibberellic acid (GA3 ) slightly delayed senescence in some experiments, but in other experiments it was without effect, and gibberellin inhibitors [ancymidol or 4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate (AMO-1618)] were ineffective as well. Salicylic acid (SA) also had no effect. Ethylene, auxins, GA3 and SA affected flower opening, therefore did reach the flower cells. Jasmonates delayed senescence by about 2.0 days. Similarly, cytokinins delayed senescence by about 1.5-2.0 days. Antagonists of the phosphatidylinositol signal transduction pathway (lithium), calcium channels (niguldipine and verapamil), calmodulin action [fluphenazine, trifluoroperazine, phenoxybenzamide and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide hydrochloride (W-7)] or protein kinase activity [1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8) and N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9)] had no effect on senescence, indicating no role of a few common signal transduction pathways relating to hormone effects on senescence. The results indicate that tepal senescence in Iris cv. Blue Magic is not regulated by endogenous ethylene, auxin, gibberellins or SA. A role of ABA can at present not be excluded. The data suggest the hypothesis that cytokinins and jasmonates are among the natural regulators.

Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca2+ ions. These two lobes have differing affinities for Ca2+, and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca2+ binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles.

Relationship between cyclic AMP‐dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa

In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)-protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP-induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP-induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) and CaCl(2) at 38.5°C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl(2) were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS-induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50-100 nM) coincidently promoted hyperactivation and cAMP-induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl(2). Moreover, the addition of W-7 (a calmodulin antagonist, 2-4 µM) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl(2), independently of protein tyrosine phosphorylation. These findings indicate that cAMP-induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP-induced protein tyrosine phosphorylation.

Electromagnetic fields instantaneously modulate nitric oxide signaling in challenged biological systems

This study shows that a non-thermal pulse-modulated RF signal (PRF), configured to modulate calmodulin (CaM) activation via acceleration of Ca2+ binding kinetics, produced an immediate nearly 3-fold increase in nitric oxide (NO) from dopaminergic MN9D cultures (P<0.001). NO was measured electrochemically in real-time using a NO selective membrane electrode, which showed the PRF effect occurred within the first seconds after lipopolysaccharide (LPS) challenge. Further support that the site of action of PRF involves CaM is provided in human fibroblast cultures challenged with low serum and exposed for 15min to the identical PRF signal. In this case a CaM antagonist W-7 could be added to the culture 3h prior to PRF exposure. Those results showed the PRF signal produced nearly a two-fold increase in NO, which could be blocked by W-7 (P<0.001). To the authors’ knowledge this is the first report of a real-time effect of non-thermal electromagnetic fields (EMF) on NO release from challenged cells. The results provide mechanistic support for the many reported bioeffects of EMF in which NO plays a role. Thus, in a typical clinical application for acute post operative pain, or chronic pain from, e.g., osteoarthritis, EMF therapy could be employed to modulate the dynamics of NO via Ca/CaM-dependent constitutive nitric oxide synthase (cNOS) in the target tissue. This, in turn, would modulate the dynamics of the signaling pathways the body uses in response to the various phases of healing after physical or chemical insult or injury.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.

Presynaptic kainate receptor‐mediated facilitation of glutamate release involves Ca2+–calmodulin at mossy fiber–CA3 synapses

Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity.

Calmodulin activation of Aurora-A kinase (AURKA) is required during ciliary disassembly and in mitosis

The centrosomal Aurora-A kinase (AURKA) regulates mitotic progression, and overexpression and hyperactivation of AURKA commonly promotes genomic instability in many tumors. Although most studies of AURKA focus on its role in mitosis, some recent work identified unexpected nonmitotic activities of AURKA. Among these, a role for basal body-localized AURKA in regulating ciliary disassembly in interphase cells has highlighted a role in regulating cellular responsiveness to growth factors and mechanical cues. The mechanism of AURKA activation involves interactions with multiple partner proteins and is not well understood, particularly in interphase cells. We show here that AURKA activation at the basal body in ciliary disassembly requires interactions with Ca(2+) and calmodulin (CaM) and that Ca(2+)/CaM are important mediators of the ciliary disassembly process. We also show that Ca(2+)/CaM binding is required for AURKA activation in mitosis and that inhibition of CaM activity reduces interaction between AURKA and its activator, NEDD9. Finally, mutated derivatives of AURKA impaired for CaM binding and/or CaM-dependent activation cause defects in mitotic progression, cytokinesis, and ciliary resorption. These results define Ca(2+)/CaM as important regulators of AURKA activation in mitotic and nonmitotic signaling.