Calmodulin Affinity Research Articles

Mg2+ Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2

Mg2+ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2+ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2+ on spontaneous Ca2+ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2+]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2+], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2+ load. To test whether Mg2+ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2+ and decreased at both 0.1 mM and 3 mM Mg2+. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2+ decreases CaSpF and increases SR Ca2+ load. As [Mg2+] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2+] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening.

Calmodulin limits pathogenic Na+ channel persistent current.

Increased "persistent" current, caused by delayed inactivation, through voltage-gated Na+ (NaV) channels leads to cardiac arrhythmias or epilepsy. The underlying molecular contributors to these inactivation defects are poorly understood. Here, we show that calmodulin (CaM) binding to multiple sites within NaV channel intracellular C-terminal domains (CTDs) limits persistent Na+ current and accelerates inactivation across the NaV family. Arrhythmia or epilepsy mutations located in NaV1.5 or NaV1.2 channel CTDs, respectively, reduce CaM binding either directly or by interfering with CTD-CTD interchannel interactions. Boosting the availability of CaM, thus shifting its binding equilibrium, restores wild-type (WT)-like inactivation in mutant NaV1.5 and NaV1.2 channels and likewise diminishes the comparatively large persistent Na+ current through WT NaV1.6, whose CTD displays relatively low CaM affinity. In cerebellar Purkinje neurons, in which NaV1.6 promotes a large physiological persistent Na+ current, increased CaM diminishes the persistent Na+ current, suggesting that the endogenous, comparatively weak affinity of NaV1.6 for apoCaM is important for physiological persistent current.

Protein-Protein Interaction: Tandem Affinity Purification in Bacteria.

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.

Calcium-dependent binding of Myc to calmodulin.

The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.

The chemosensitizing agent lubeluzole binds calmodulin and inhibits Ca2+/calmodulin-dependent kinase II

An affinity capillary electrophoresis (ACE) method to estimate apparent dissociation constants between bovine brain calmodulin (CaM) and non-peptidic ligands was developed. The method was validated reproducing the dissociation constants of a number of well-known CaM ligands. In particular, the potent antagonist 125-C9 was ad hoc synthesized through an improved synthetic procedure. The ACE method was successfully applied to verify CaM affinity for lubeluzole, a well-known neuroprotective agent recently proved useful to potentiate the activity of anti-cancer drugs. Lubeluzole was slightly less potent than 125-C9 (Kd = 2.9 ± 0.7 and 0.47 ± 0.06 μM, respectively) and displayed Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition (IC50 = 40 ± 1 μM). Possible binding modes of lubeluzole to CaM were explored by docking studies based on the X-ray crystal structures of several trifluoperazine-CaM complexes. An estimated dissociation constant in good agreement with the experimental one was found and the main aminoacidic residues and interactions contributing to complex formation were highlighted. The possibility that interference with Ca2+ pathways may contribute to the previously observed chemosensitizing effects of lubeluzole on human ovarian adenocarcinoma and lung carcinoma cells are discussed.

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin Variants in Long QT Syndrome and Functional Characterization of a Novel Long QT Syndrome-Associated Calmodulin Missense Variant, E141G.

Calmodulin (CaM) is encoded by 3 genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca(2+) and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P<0.0001). When the clinical sequelae of these 5 CaM-positive cases were compared with the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 10 months, an average corrected QT interval of 676 ms, and a high prevalence of cardiac arrest. Functional characterization of 1 novel variant, E141G-CaM, revealed an 11-fold reduction in Ca(2+)-binding affinity and a functionally dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

Centrin 4 is a Binding Partner of Rat EAG1 K+ Channels

The ether-a-go-go (Eag) potassium (K+) channel belongs to the superfamily of voltage-gated K+ channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was centrin 4, which is an EF-hand calcium-binding protein with four putative calciumion-binding sites. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with centrin was mediated by the carboxyl-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and brain lysates. Centrin 4 and calmodulin are closely related four-EF-hand calcium-binding proteins. The binding affinity of calmodulin to rEag1 was notably enhanced in the presence of calcium. In contrast, the interaction between centrin 4 and rEag1 appeared to be calcium-independent. We therefore propose that centrin 4 may serve as a constitutive binding-partner of rEag1.

The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form.

Abstract 13700: High Affinity CaM, but Not Normal Affinity CaM Can Prevent Arrhythmias in CPVT Which Mutation is Located at CaM-like Domain of RyR2

Introduction: We have been previously reported that calmodulin (CaM) binding affinity to RyR2 decreases upon beta stimulation in catecholaminergic polymorphic ventricular tachycardia (CPVT) type knock-in mice with mutation at central domain (R2474S-KI). Meanwhile, amino acid residues from 4026 to 4172 of RyR2 have EF hand motifs and are called CaM-like domain (CaMLD). Some of the human CPVT mutations were reported in this CaMLD. In the present study we investigated the molecular mechanism of arrhythmogenesis of CPVT with mutation at CaMLD using N4103K KI mice. Methods and Results: VT and/or Vf were frequently observed after 120 mg/kg caffeine plus 1 mg/kg epinephrine infusion in KI mice, but not in WT mice (WT: 0%, KI: 43%). CaM binding affinity to RyR2 was assessed by incorporation of exogenous CaM labeled with HiLyte Fluor® in isolated saponin-permeabilized cardiomyocytes. The binding affinity of CaM decreased in KI cells (Kd: 161 nM) compared with WT cells (Kd: 89 nM). Addition of 1μM cAMP did not change the affinity of CaM to RyR2 in both KI and WT cells, while it decreased the affinity in R2474S-KI. Frequency of the Ca 2+ sparks was measured in isolated saponin-permeabilized cells. The Ca 2+ spark frequency (SpF: s -1 100μm -1 ) was much higher in KI than in WT cells {KI (n=79): 21.5±6.3; WT (n=34): 10.6±4.1; p&lt;0.01}. Addition of 100 nM CaM to KI cells did not decrease SpF {(n=87): 22.4±6.5}, however, significant decrease was observed by HA-CaM (modified CaM which has higher affinity to RyR2). Spontaneous Ca 2+ transient (SCaT) was measured during sequential pacing from 1 to 5 Hz in isolated intact cardiomyocytes. The SCaT was rarely observed in WT cells even with 10 nM isoproterenol and 5 Hz pacing, whereas that was observed in 33% of KI cells. Incorporation of CaM to intact KI cells using protein delivery kit (BioPORTER®) did not decrease incidence of SCaT (34% of KI cells), however, significantly decreased by HA-CaM incorporation. Conclusions: Different from R2474S-KI mice, CaM binding affinity to RyR2 is decreased without beta stimulation in KI mice with CaMLD mutation. Conformational change of CaMLD might affect CaM-binding domain (CaMBD) and decrease the affinity of CaM to CaMBD. The CaMLD-CaMBD interaction may be a novel therapeutic target of lethal arrhythmias.

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K+ channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca2+). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction.

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.

CaMKII-Dependent Phosphorylation of RyR2 Causes Domain Unzipping and Reduced Calmodulin Binding, But Dantrolene Reverses These Effects

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). CaMKII-dependent RyR2 phosphorylation (at S2814) is thought to be a critical promoter of leaky RyR2 in HF. A conformational change in RyR2 (“domain unzipping”) observed in HF can also increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and the CaM affinity for RyR2 is reduced in HF. Further, we have shown reciprocal negative coupling between CaM binding to RyR2 and accessibility of the unzipping RyR peptide DPc10 (Oda et al. 2013). That is, unzipping with DPc10 reduced CaM binding and increased Ca leak, whereas CaM quieted leak and inhibited DPc10 access. Here, we tested the hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM affinity. We either activated endogenous myocyte CaMKII or used knock-in mice expressing non-phosphorylatable RyR2-S2814A or phosphomimetic RyR2-S2814D. We used FRET to directly detect binding of fluorescently labeled DPc10, FKBP12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In phosphomimetic S2814D vs. S2814A myocytes, CaM-RyR2 affinity was reduced 3-fold (Kd= 72 ± 9 nM vs. 20 ± 2 nM), but FRETmax was unaltered. Access of DPc10 was also 2-fold faster in S2814D versus S2814A myocytes. Dantrolene (1 μM), which stabilizes the zipped conformation, restored CaM-RyR2 binding affinity in S2814D, slowed DPc10 wash-in, and lowered Bmax. Forced RyR2-S2814D saturation with 500 nM CaM also slowed DPc10 wash-in and reduced Bmax. We conclude that RyR2 phosphorylation by CaMKII causes pathological conformation changes (unzipping) that reduce RyR2-CaM affinity and ability to quiet SR Ca leak. Dantrolene effectively reverses these effects.

Binding Affinity of Calmodulin to Cardiac Ryanodine Receptor, as the Novel Therapeutic Target in the Pressure-overloaded Heart Failure

Binding Affinity of Calmodulin to Cardiac Ryanodine Receptor, as the Novel Therapeutic Target in the Pressure-overloaded Heart Failure

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.

Determinants of Preferential Binding of Apo Calmodulin to the IQ Motif of Neuronal Sodium Channel NaV1.2

The neuronal voltage-gated sodium channel (NaV1.2) is regulated by calmodulin (CaM), a highly conserved, ubiquitous eukaryotic protein that mediates many calcium-triggered signaling events. Fast inactivation of the channel depends on CaM-mediated feedback transduction of calcium flux during the repolarization phase of an action potential. CaM binds to an intracellular loop (the III-IV linker) and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail of the channel. The NaV1.2 IQ motif binds only to the C-domain of CaM with high affinity to both its calcium-free (apo) and calcium-saturated states. However, the IQ motif binds more favorably to apo CaM than to calcium-saturated CaM. To determine the molecular basis for this calcium-dependent difference in association, mutational perturbations of residues in the NaV1.2 IQ motif were designed to disrupt close contacts observed in our solution (NMR) structure of the semi-open C-domain of apo-CaM bound to the IQ motif (2KXW.pdb). The contributions of these residues to binding energetics were determined by monitoring CaM-induced disruption of FRET in biosensors containing wild-type or mutant sequences of the IQ motif bracketed by auto-fluorescent proteins YFP and CFP. All mutations lowered affinity for calcium-saturated CaM, but they had uniformly more deleterious effects on the binding of apo CaM. Furthermore, the decrease in affinity for apo CaM caused by loss of the Ile-Gln pair was 30-fold greater than that observed for loss of the Tyr-Tyr pair. Thus, the energy of interaction between the NaV1.2 IQ motif and semi-open apo CaM is not accounted for primarily by the classical “aromatic anchors” that dominate interactions of calcium-saturated CaM with its target sequences in kinases, receptors and other channels. Support: NIH R01 GM57001, Carver Charitable Trust Grant 01-224.

Positive Cooperativity and T286 Autophosphorylation is Observed in a Dimeric Mutant of Calcium-Calmodulin Dependent Protein Kinase II (CaMKII)

CaMKII is a multimeric enzyme that regulates long-term potentiation in the hippocampus. The assembly and organization of 8-14 subunits in a holoenzyme is thought to be required for transduction of calcium spike frequencies, cooperative binding to calmodulin, persistent activation by T286 autophosphorylation, and translocation into synaptic spines. CaMKII assembles into a holoenzyme by virtue of its unique C-terminal association domain (AD). ADs form a central hub-like structure from which regulatory and catalytic domains project. Each AD interacts tightly with three other subunits, two laterally and one transversely, to form a stable core composed of two stacked rings. FRET and analytical centrifugation has indicated that individual catalytic domains can form pairs by virtue of low affinity binding sites. But catalytic domains of the CaMKII mutant lacking AD do not form pairs in physiological conditions. We hypothesize that catalytic domain pairing is the fundamental structure that allows positive cooperativity and T286 autophosphorylation. Furthermore we postulate that high affinity AD core interactions lead to a high local concentration of catalytic domains that allows low-affinity catalytic domain pairing. To test this hypothesis, we mutated the lateral surface of the CaMKIIα AD. When expressed in HEK293 cells, the mutation generated a paired dimeric CaMKIIα. In support of the hypothesis, our study shows that the dimeric enzyme has the same affinity for calmodulin, a similar Hill coefficient for enzymatic activity, and T286 autophosphorylation comparable to that observed in the wild type holoenzyme. In contrast, the monomeric mutant CaMKII lacking AD has lower affinity, no cooperativity, and reduced T286 autophosphorylation. While catalytic domain pairing was observed in the dimeric mutant, biophysical analysis suggests that the nature of the pairing might be altered.

Biochemical Assays of CaM and PiP2 Interactions with KCNQ Channel Domains

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are involved in controlling the gating of KCNQ ("M-type") K+ channels. It is well established that CaM has affinity for both the A and B helices of the KCNQ proximal C-terminus, and previous work indicates that CaM may be constitutively bound to the B helix. KCNQ channels are also suggested to contain multiple PIP2-binding sites, including a region of basic residues between the A and B helices (A-B linker) and a basic region at the C-terminal junction with the S6 transmembrane domain (S6Jx). Using a novel approach to understand the mechanisms controlling the modulation of KCNQ channels, we compared the biochemical affinities of PIP2 and CaM for these corresponding domains. Isothermal titration calorimetry (ITC) revealed a high affinity of Ca2+/CaM for a peptide corresponding to the KCNQ3 B helix (Kd ∼82 nM), whereas Ca2+/CaM bound the A helix with a much higher Kd (∼2 μM). The interactions between apo-CaM and the A or B helix peptide were not strong enough to obtain Kd values. Analytical ultracentrifugation results indicated that PIP2 directly bound to the S6Jx peptide, whereas its interaction with the A helix-B helix linker peptide is, as yet, indeterminate. These findings support the idea that CaM binds both the A and B helices of the KCNQ C-terminal domain in a Ca2+-dependent manner. Since the S6Jx domain is known to be close to the cell membrane by many structural studies of K+ channels, these data also suggest that only a very small conformational change in the C-termini of KCNQ channels is required to alter its binding to membrane PIP2. We will present more biochemical results that should help define the structural and molecular mechanism of PIP2 and CaM actions on KCNQ channels.