Calmodulin Target Research Articles

Oxidation of Methionine 77 in Calmodulin Alters Mouse Growth and Behavior.

Methionine 77 in calmodulin can be stereospecifically oxidized to methionine sulfoxide by mammalian methionine sulfoxide reductase A. Whether this has in vivo significance is unknown. We therefore created a mutant mouse in which wild type calmodulin-1 was replaced by a calmodulin containing a mimic of methionine sulfoxide at residue 77. Total calmodulin levels were unchanged in the homozygous M77Q mutant, which is viable and fertile. No differences were observed on learning tests, including the Morris water maze and associative learning. Cardiac stress test results were also the same for mutant and wild type mice. However, young male and female mice were 20% smaller than wild type mice, although food intake was normal for their weight. Young M77Q mice were notably more active and exploratory than wild type mice. This behavior difference was objectively documented on the treadmill and open field tests. The mutant mice ran 20% longer on the treadmill than controls and in the open field test, the mutant mice explored more than controls and exhibited reduced anxiety. These phenotypic differences bore a similarity to those observed in mice lacking calcium/calmodulin kinase IIα (CaMKIIα). We then showed that MetO77 calmodulin was less effective in activating CaMKIIα than wild type calmodulin. Thus, characterization of the phenotype of a mouse expressing a constitutively active mimic of calmodulin led to the identification of the first calmodulin target that can be differentially regulated by the oxidation state of Met77. We conclude that reversible oxidation of methionine 77 in calmodulin by MSRA has the potential to regulate cellular function.

Investigation of the structure and dynamic of calmodulin-nitric oxide synthase complexes using NMR spectroscopy.

NMR spectroscopy allows for the determination of high resolution structures, as well as being an efficient method for studying the dynamics of protein-protein and protein-peptide complexes. 15N relaxation and H/D exchange experiments allow for the analysis of these structural dynamics at a residue specific level. Calmodulin (CaM) is a small cytosolic Ca2+ binding protein that serves as a control element for many enzymes. An important target of CaM are the nitric oxide synthase (NOS) enzymes that play a major role in a number of key physiological and pathological processes. Studies have shown CaM facilitates a conformational shift in NOS allowing for efficient electron transfer through a process thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. This review highlights recent work performed on the CaM-NOS complexes using NMR spectroscopy and shows remarkable differences in the dynamic properties of CaM-NOS complexes at physiologically relevant Ca2+ concentrations. It also shows key structural changes that affect the activity of NOS when interacting with apoCaM mutants and NOS posttranslational modifications are present.

Functions of IQD proteins as hubs in cellular calcium and auxin signaling: A toolbox for shape formation and tissue-specification in plants?

ABSTRACTCalcium (Ca2+) ions play pivotal roles as second messengers in intracellular signal transduction, and coordinate many biological processes. Changes in intracellular Ca2+ levels are perceived by Ca2+ sensors such as calmodulin (CaM) and CaM-like (CML) proteins, which transduce Ca2+ signals into cellular responses by regulation of diverse target proteins. Insights into molecular functions of CaM targets are thus essential to understand the molecular and cellular basis of Ca2+ signaling. During the last decade, IQ67-domain (IQD) proteins emerged as the largest class of CaM targets in plants with mostly unknown functions. In the March issue of Plant Physiology, we presented the first comprehensive characterization of the 33-membered IQD family in Arabidopsis thaliana. We showed, by analysis of the subcellular localization of translational green fluorescent protein (GFP) fusion proteins, that most IQD members label microtubules (MTs), and additionally often localize to the cell nucleus or to membranes, where they recruit CaM Ca2+ sensors. Important functions at MTs are supported by altered MT organization and plant growth in IQD gain-of-function lines. Because IQD proteins share structural hallmarks of scaffold proteins, we propose roles of IQDs in the assembly of macromolecular complexes to orchestrate Ca2+ CaM signaling from membranes to the nucleus. Interestingly, expression of several IQDs is regulated by auxin, which suggests functions of IQDs as hubs in cellular auxin and calcium signaling to regulate plant growth and development.

The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin.

IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93KK94 are important for CaM binding, and indeed, the 93EE94 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the 93KK94 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.

Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen

To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13–Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles.

Functional Rescue of Calmodulinopathy IPSC-Derived Cardiomyocytes -- a Foray into Personalized Medicine

Calmodulinopathies comprise a growing population of patients with congenital life-threatening cardiac arrhythmias, including severe long-QT syndrome (LQTS). The underlying mutations disrupt Ca2+ binding to calmodulin (CaM), implicating a myriad of Ca2+/CaM targets as potential pathogenic elements. To gain further mechanistic insight, we utilized induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), a model of congenital cardiac diseases with a patient-specific genetic background. First, we demonstrated that calmodulinopathy iPSC-CMs indeed display (1) significantly prolonged action potentials (APs), (2) increased Ca2+ transient (CaT) amplitudes, (3) delayed CaT upstrokes, and (4) drastically slower CaT decay rates -- all potential components of an arrhythmic substrate and an ideal platform for developing a treatment strategy. Next, we considered the role of the L-type Ca2+ channel (LTCC) in arrhythmogenesis within these cells. Indeed, the iPSC-CMs displayed severely diminished LTCC Ca2+/CaM-dependent inactivation (CDI), a known mechanism for AP prolongation. Finally, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only a single of three redundant CaM genes, we have devised a strategy to selectively suppress the mutant gene while sparing the wild-type counterparts. The extent of this suppression was confirmed via quantitative RNA expression profiling. Importantly, this manipulation produced a functional rescue (restoration of the AP duration) in calmodulinopathy iPSC-CMs in a dose-dependent manner. Moreover, selective knockdown of any of the three genes underlying calmodulinopathies could be achieved. This technique not only promises significant relief of LQT symptoms, but any disease manifestations in these patients as well. Thus, we have expanded the therapeutic toolkit for treatment of calmodulinopathies and initiated a path towards personalized medicine.

Neurogranin restores amyloid β-mediated synaptic transmission and long-term potentiation deficits

Amyloid β (Aβ) is widely considered one of the early causes of cognitive deficits observed in Alzheimer's disease. Many of the deficits caused by Aβ are attributed to its disruption of synaptic function represented by its blockade of long-term potentiation (LTP) and its induction of synaptic depression. Identifying pathways that reverse these synaptic deficits may open the door to new therapeutic targets. In this study, we explored the possibility that Neurogranin (Ng)—a postsynaptic calmodulin (CaM) targeting protein that enhances synaptic function—may rescue Aβ-mediated deficits in synaptic function. Our results show that Ng is able to reverse synaptic depression and LTP deficits induced by Aβ. Furthermore, Ng's restoration of synaptic transmission is through the insertion of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPARs). These restorative effects of Ng are dependent on the interaction of Ng and CaM and CaM-dependent activation of CaMKII. Overall, this study identifies a novel mechanism to rescue synaptic deficits induced by Aβ oligomers. It also suggests Ng and CaM signaling as potential therapeutic targets for Alzheimer's disease as well as important tools to further explore the pathophysiology underlying the disease.

Enhanced Linkage in the Vascular Calmodulin Network by Estrogen via a Feedforward at the G Protein‐Coupled Estrogen Receptor 1 (GPER/GPR30)

Plasma estrogen is strongly linked to cardiovascular health. Calmodulin (CaM) is required for the activities of numerous proteins but is insufficiently expressed for all its targets, leading to competition among CaM targets. It is completely unknown if estrogen modulates cardiovascular functions via the CaM network. We observed in endothelial cells that chronic estrogen treatment (CE2T) increases total and free CaM. Chronic treatment with G‐1, an agonist of the estrogen‐sensitive receptor GPER/GPR30 mimics this effect, while agonists of ERa or ERb do not. ICI182780, an ERa/ERb antagonist and GPER/GPR30 agonist, also increases CaM level. CE2T increases CaM binding to different categories of CaM targets, including the plasma membrane Ca2+‐ATPase (PMCA), eNOS, ERa, and GPER/GPR30 itself. For GPER/GPR30, CaM antagonism or CaM binding‐negating mutations in the receptor's multiple CaM‐binding domains prevent GPER/GPR30‐mediated ERK1/2 phosphorylation. For PMCA, CE2T‐induced stimulation of activity through enhanced CaM binding is masked by Src‐dependent phosphorylation. These effects sustain cytoplasmic Ca2+ for enhanced interactions between CaM and other targets. For eNOS, CE2T doubles CaM binding. Kinetic modeling using in‐cell and in vitro data allowed comparison of CE2T's promotion of eNOS point activity and NO accumulation via effects on determinants of eNOS function, including Ca2+, CaM, and phosphorylation. Our data indicate that CE2T improves endothelial functions via a feed‐forward mechanism in which CaM is upregulated through GPER/GPR30 activation, leading to enhanced CaM binding and functional linkage in the network of CaM‐binding proteins.

Conformational frustration in calmodulin-target recognition.

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.

Tuning of Structure-Function Relationships by Macromolecular Crowding

The cellular interior is crowded with high concentrations of macromolecules and small organic solutes and strict maintenance of this environment, where ∼30% of the total volume is occupied, is essential to cellular function. The volume occupied by these molecules confines the 3D space in which any particular protein can reside and is thought to modulate protein-protein interactions by affecting diffusional encounter and reaction rates. In this work, we investigate the mechanistic consequences of crowded environments on the structure-function relationship of the ubiquitous protein calmodulin (CaM). CaM is highly flexible and dynamic, making it particularly susceptible to crowded environments, and its conformational plasticity is essential for accommodating binding to its 300 identified targets. We determined that crowded environments, created with purified polymer systems and sucrose, stabilize compact conformers of CaM, reduce translational and rotational diffusion, modulate association and dissociation kinetics with a calmodulin-binding target, and control rates of conformational transition. Most notably, these effects are determined by the size and concentration of the crowding reagent indicating that the composition of the environment differentially tunes CaM structure, dynamics, and target association kinetics. Our results are shown in a general kinetic framework where there are consequences for target selectivity and cellular signaling.

The Role of Calmodulin and Related Proteins in Plant Cell Function: An Ever-Thickening Plot

Stimuli-induced fluctuations in intracellular free calcium (Ca2+) serve as secondary messenger signals that regulate diverse biochemical processes in eukaryotic cells, such as developmental transitions and responses to biotic and abiotic stresses. Stimuli-specific Ca2+ signals are manifested as spatially and temporally defined differential Ca2+ signatures that are sensed, decoded, and transduced to elicit distal responses via an array of Ca2+ binding proteins (CBPs) that function as intracellular Ca2+ sensors. Calmodulin (CaM), the most important eukaryotic CBP, senses and responds to fluctuations in intracellular Ca2+ levels by binding to this ubiquitous second messenger, and transducing given Ca2+ signatures that differentially activate distal effector (target) proteins regulating a broad range of biochemical responses. Ca2+/CaM targets include an increasing number of proteins whose functions continue to be elucidated. Hundreds of reports have highlighted the importance of CaM, and other CBPs, in the transduction of Ca2+-mediated signals involved in transcriptional regulation, protein phosphorylation/dephosphorylation, and metabolic shifts. Other Ca2+-binding proteins are known to play significant functional roles in plant cells as well. This review is primarily focused on the role of CaM in some key plant processes, and discusses recent advances in understanding the pivotal role of CaM in an ever-increasing number of plant cell functions and biochemical responses. We also discuss recent work highlighting the emerging importance of CaM in nuclear and organellar signaling.

Role of calcium in membrane interactions by PI(4,5)P₂-binding proteins.

Ca²⁺ and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P₂ has been shown to be tightly regulated and dependent on the presence of Ca²⁺, with co-operative binding in some cases. In these proteins, PI(4,5)P₂ and Ca²⁺ binding typically occurs at different binding sites. In addition, several PI(4,5)P₂-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P₂ for protein interaction, translating Ca²⁺ transient microdomains into variations of PI(4,5)P₂ lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P₂-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P₂.

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin.

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term = 2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins.

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM.

Structural insights into calmodulin/Munc13 interaction.

Munc13 proteins are essential presynaptic regulators that mediate synaptic vesicle priming and play a role in the regulation of neuronal short-term synaptic plasticity. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. Here, we summarize recent results on the investigation of the CaM/Munc13 interaction. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we showed that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrated that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK, indicating unique features of the Munc13 CaM binding motif.

Solution Structure of Calmodulin Bound to the Target Peptide of Endothelial Nitric Oxide Synthase Phosphorylated at Thr495

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α-helix. The calcium affinity of the CaM-target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue.

Mapping the sevoflurane-binding sites of calmodulin.

General anesthetics, with sevoflurane (SF) being the first choice inhalational anesthetic agent, provide reversible, broad depressor effects on the nervous system yet have a narrow margin of safety. As characterization of low-affinity binding interactions of volatile substances is exceptionally challenging with the existing methods, none of the numerous cellular targets proposed as chief protagonists in anesthesia could yet be confirmed. The recognition that most critical functions modulated by volatile anesthetics are under the control of intracellular Ca2+ concentration, which in turn is primarily regulated by calmodulin (CaM), motivated us for characterization of the SF–CaM interaction. Solution NMR (Nuclear Magnetic Resonance) spectroscopy was used to identify SF-binding sites using chemical shift displacement, NOESY and heteronuclear Overhauser enhancement spectroscopy (HOESY) experiments. Binding affinities were measured using ITC (isothermal titration calorimetry). SF binds to both lobes of (Ca2+)4-CaM with low mmol/L affinity whereas no interaction was observed in the absence of Ca2+. SF does not affect the calcium binding of CaM. The structurally closely related SF and isoflurane are shown to bind to the same clefts. The SF-binding clefts overlap with the binding sites of physiologically relevant ion channels and bioactive small molecules, but the binding affinity suggests it could only interfere with very weak CaM targets.

Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A

N-methyl-d-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875–1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca2+/calmodulin binds GluN2A with high affinity (5.2±2.4nM) in vitro. Direct interaction of Ca2+/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca2+/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca2+/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.