Characterization of the C-terminal half of human juvenile myoclonic epilepsy protein EFHC1: dimer formation blocks Ca2+ and Mg2+ binding to its functional EF-hand

Human EFHC1 is a member of the EF-hand superfamily of Ca²⁺-binding proteins with three DM10 domains of unclear function. Point mutations in the EFHC1 gene are related to juvenile myoclonic epilepsy, a fairly common idiopathic generalized epilepsy. Here, we report the first structural and thermodynamic analyses of the EFHC1C-terminus (residues 403-640; named EFHC1C), comprising the last DM10 domain and the EF-hand motif. Circular dichroism spectroscopy revealed that the secondary structure of EFHC1C is composed by 34% of α-helices and 17% of β-strands. Size exclusion chromatography and mass spectrometry showed that under oxidizing condition EFHC1C dimerizes through the formation of disulfide bond. Tandem mass spectrometry (MS/MS) analysis of peptides generated by trypsin digestion suggests that the Cys575 is involved in intermolecular S-S bond. In addition, DTNB assay showed that each reduced EFHC1C molecule has one accessible free thiol. Isothermal titration calorimetry (ITC) showed that while the interaction between Ca²⁺ and EFHC1C is enthalpically driven (ΔH = −58.6 to −67 kJ/mol and TΔS = −22.5 to −31 kJ/mol) the interaction between Mg²⁺ and EFHC1C involves an entropic gain, and is ∼5 times less enthalpically favorable (ΔH = −11.7 to −14 kJ/mol and TΔS = 21.9 to 19 kJ/mol) than for Ca²⁺ binding. It was also found that under reducing condition Ca²⁺ or Mg²⁺ ions bind to EFHC1C in a 1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFHC1C dimerization blocks Ca²⁺ and Mg²⁺ binding.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Gap junction-mediated spontaneous Ca waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca²⁺ waves. These waves were unaffected by tetrodotoxin (1 μM), but blocked by removal of extracellular Ca²⁺, or addition of non-specific Ca²⁺ channel inhibitors (Cd²⁺ (0.1 mM) or Ni²⁺ (1 mM)). Combined application of antagonists of NMDA receptors (AP5; 100 μM), AMPA/kainate receptors (NBQX; 20 μM), nicotinic AChR receptors (hexamethonium; 100 μM) or inotropic purinoceptors (brilliant blue; 100 nM) was also without effect. However, Ca²⁺ waves were fully prevented by carbenoxolone (200 μM), halothane (3 mM) or niflumic acid (100 μM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd²⁺, Ni²⁺ or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca²⁺ waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.     

Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca²⁺) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca²⁺, we characterized Ca²⁺ homeostasis in P. aeruginosa PAO1 cells. By using Ca²⁺-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca²⁺ ([Ca²⁺]_in) is 0.14 ± 0.05 μM. In response to external Ca²⁺, the [Ca²⁺]_in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca²⁺ modulated this response. Treatment with inhibitors known to affect Ca²⁺ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca²⁺]_in response, suggesting the importance of the corresponding mechanisms in Ca²⁺ homeostasis. To identify Ca²⁺ transporters maintaining this homeostasis, bioinformatic and LC–MS/MS-based membrane proteomic analyses were used. [Ca²⁺]_in homeostasis was monitored for seven Ca²⁺-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca²⁺ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca²⁺-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca²⁺.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg²⁺, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg²⁺ and Ca²⁺ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k_OH) of 4.1 × 10⁻⁴ M⁻¹s⁻¹ and 3.7 × 10⁻⁴ M⁻¹s⁻¹ in the presence of 0.30 M Mg²⁺ and Ca²⁺, respectively, compared to 8.3 × 10⁻⁷ M⁻¹s⁻¹ in the absence of divalent metal ion. Examining the dependence of k_OH on [M²⁺] at 50 °C indicates different kinetic mechanisms with Mg²⁺ utilizing a single ion mechanism and Ca²⁺ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k_OH values reveals a single Mg²⁺ catalyzes the reaction by 1800-fold whereas a single Ca²⁺ ion catalyzes the reaction by only 90-fold. The second Ca²⁺ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg²⁺ and Ca²⁺.     

Modeling local and global intracellular calcium responses mediated by diffusely distributed inositol 1,4,5-trisphosphate receptors

Considerable insight into intracellular Ca²⁺ responses has been obtained through the development of whole cell models that are based on molecular mechanisms, e.g., single channel kinetics of the inositol 1,4,5-trisphosphate (IP₃) receptor Ca²⁺ channel. However, a limitation of most whole cell models to date is the assumption that IP₃ receptor Ca²⁺ channels (IP₃Rs) are globally coupled by a “continuously stirred” bulk cytosolic [Ca²⁺], when in fact open IP₃Rs experience elevated “domain” Ca²⁺ concentrations. Here we present a 2N+2-compartment whole cell model of local and global Ca²⁺ responses mediated by N=100,000 diffusely distributed IP₃Rs, each represented by a four-state Markov chain. Two of these compartments correspond to bulk cytosolic and luminal Ca²⁺ concentrations, and the remaining 2N compartments represent time-dependent cytosolic and luminal Ca²⁺ domains associated with each IP₃R. Using this Monte Carlo model as a starting point, we present an alternative formulation that solves a system of advection-reaction equations for the probability density of cytosolic and luminal domain [Ca²⁺] jointly distributed with IP₃R state. When these equations are coupled to ordinary differential equations for the bulk cytosolic and luminal [Ca²⁺], a realistic but minimal model of whole cell Ca²⁺ dynamics is produced that accounts for the influence of local Ca²⁺ signaling on channel gating and global Ca²⁺ responses. The probability density approach is benchmarked and validated by comparison to Monte Carlo simulations, and the two methods are shown to agree when the number of Ca²⁺ channels is large (i.e., physiologically realistic). Using the probability density approach, we show that the time scale of Ca²⁺ domain formation and collapse (both cytosolic and luminal) may influence global Ca²⁺ oscillations, and we derive two reduced models of global Ca²⁺ dynamics that account for the influence of local Ca²⁺ signaling on global Ca²⁺ dynamics when there is a separation of time scales between the stochastic gating of IP₃Rs and the dynamics of domain Ca²⁺.     

Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes

Aims

Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).

Methods and results

Transmembrane potentials were recorded in whole cell current mode before and after pacing (1–5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor® in saponin-permeabilized myocytes. In the presence of cAMP (1 μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140–400 nM), but not in WT cells (Kd: 110–120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca²⁺ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca²⁺ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM] = ∼240 nM, cytosolic [GSH-CaM] = ∼230 nM) on the frequency of spontaneous Ca²⁺ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P < 0.01 vs CaM).

Conclusions

Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca²⁺ leak, delayed afterdepolarization, triggered activity, Ca²⁺ spark frequency, spontaneous Ca²⁺ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.     

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca²⁺ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca²⁺-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca²⁺]_i measurement in GFP expressing cells. The indicators bind Ca²⁺ with either high (K_d = 0.49 ± 0.07 μM; ACR-1) or low affinity (K_d = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn²⁺ (K_d = 0.38 ± 0.02 nM) or Mg²⁺ (K_d ∼ 5 mM) slightly raises and binding Co²⁺ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pK_a = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.     

Electrophoretic mobility of sarcoplasmic reticulum vesicles is determined by amino acids of A + P + N domains of Ca-ATPase

Establishing the origin of electrophoretic mobility of sarcoplasmic reticulum (SR) vesicles is the primary goal of this work. It was found that the electrophoretic mobility originates from ionizable amino acids of cytoplasmic domains of the Ca²⁺-ATPase, the calcium pump of SR. The mobility was measured at pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in the region of ionic strength from 0.05 to 0.2 M. Mobility measurements were supplemented by studies of SR vesicles by photoelectron microscopy. The median diameter of SR vesicles was 260 nm. Ca²⁺-ATPases were not resolved. The mobility data were standardized by interpolation to a reference ionic strength of 0.1 M. The mobility of the SR vesicles is determined by the charge of the Ca²⁺-ATPase. It is due to the ionizable amino acids selected from the amino acid sequence of SERCA1a Ca²⁺-ATPase. The pH dependence of charge residing in various domains of Ca²⁺-ATPase was computed using pKa values in free water. The charge correlated with measured mobility. It was shown that a linear relationship exists between the mobility of the SR vesicles, μ, and the total computed charge, Q, on three cytoplasmic domains of Ca²⁺-ATPase: A, P, and N. It is given by μ = α + β Q where the fitted values β = (0.043 ± 0.002) × 10⁻⁸ m² V⁻¹ s⁻¹ e⁻¹ and α = (0.16 ± 0.02) × 10⁻⁸ m² V⁻¹ s⁻¹. Since β and α values do not change from pH 4 to pH 9, one concludes that the hydrodynamic friction of the cytoplasmic domains of SR is independent of their charge.     

Interaction of calcium with the human divalent metal-ion transporter-1

Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interaction is not understood. We tested the hypothesis that divalent metal-ion transporter-1 (DMT1)—the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border—is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate ⁴⁵Ca²⁺ uptake. Instead, we found that Ca²⁺ blocked the Fe²⁺-evoked currents and inhibited ⁵⁵Fe²⁺ uptake in a noncompetitive manner (K_i ≈ 20 mM). The mechanism of inhibition was independent of voltage and did not involve intracellular Ca²⁺ signaling. The alkaline-earth metal ions Ba²⁺, Sr²⁺, and Mg²⁺ also inhibited DMT1-mediated iron-transport activity. We conclude that Ca²⁺ is a low-affinity noncompetitive inhibitor—but not a transported substrate—of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability.     

How calcium controls microtubule anisotropic phase formation in the presence of microtubule-associated proteins in vitro

Here we show a new effect of Ca²⁺ on microtubule morphology: Ca²⁺ can cause smooth curving of microtubules in the presence of microtubule-associated proteins (MAPs). In vitro, microtubules self-organize, forming complex dissipative structures. Such structures may be strongly affected by relatively weak external factors. A factor such as Ca²⁺ potentially influences spatiotemporal patterns of microtubule assembly, but the dynamics are unclear. We tested Ca²⁺ effects on microtuble formation. Using EM, microtubule length, curvature, and alignment and were measured in two systems: 2 mg/ml microtubule protein containing MAPs and 1 mM EGTA with and without 1 mM Ca²⁺. The two systems were then tested using light scattering. In low Ca²⁺, a birefringent microtubular pattern is seen, increasing with polymerization. When 1 mM Ca²⁺ is added to the solution. anisotropic phase is prevented without microtubule disruption. This demonstrates an additional mechanism by which Ca²⁺ can alter the dynamics and morphology of microtules.     

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 ± 2.4 nM) 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.     

Rosiglitazone transiently disturbs calcium homeostasis in monocytic cells

The PPARγ agonist Rosiglitazone exerts anti-hyperglycaemic effects by regulating the long-term expression of genes involved in metabolism, differentiation and inflammation. In the present study, Rosiglitazone treatment rapidly inhibited (5-30 min) the ER Ca²⁺ ATPase SERCA2b in monocytic cells (IC₅₀ = 1.88 μM; p < 0.05), thereby disrupting short-term Ca²⁺ homeostasis (resting [Ca²⁺]_cyto = 121.2 ± 2.9% basal within 1 h; p < 0.05). However, extended Rosiglitazone treatment (72 h) induced dose-dependent SERCA2b up-regulation, and restored calcium homeostasis, in monocytic cells (SERCA2b mRNA: 138.7 ± 5.7% basal (1 μM)/215.0 ± 30.9% basal (10 μM); resting [Ca²⁺]_cyto = 97.3 ± 8.3% basal (10 μM)). As unfavourable cardiovascular outcomes, possibly related to disrupted cellular Ca²⁺ homeostasis, have been linked to Rosiglitazone, this effect may be of clinical interest. In contrast, in PPRE-luciferase reporter-gene assays, Rosiglitazone induced non-dose-dependent PPARγ-dependent effects (1 μM: 152.5 ± 4.9% basal; 10 μM: 136.1 ± 5.1% basal (p < 0.05 for 1 μM vs. 10 μM)). Thus, we conclude that Rosiglitazone can exert PPARγ-independent non-genomic effects, such as the SERCA2b inhibition seen here, but that long-term Rosiglitazone treatment did not perturb resting [Ca]_cyto in this study.