Physiological Calcium Concentrations Research Articles

Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m) under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum) concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD) varieties. We establish (a) that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b) that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c) that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d) that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT), resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

Diaphragm Fiber Strength Is Reduced in Critically Ill Patients and Restored by a Troponin Activator

Diaphragm Fiber Strength Is Reduced in Critically Ill Patients and Restored by a Troponin Activator

Regulation of endothelial nitric oxide synthase phosphorylated at Thr495 by interaction with calmodulin (769.8)

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. Calmodulin (CaM), a small acidic calcium binding protein, 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 electron transfer 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 of eNOS that contains a phosphorylated threonine corresponding to Thr495 in order 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 due to phosphorylation and this leads to weaker binding under low physiological calcium concentrations. This study provides an explanation for the reduced NO production by eNOS carrying a phosphorylated Thr495 residue.Grant Funding Source: National Science and Engineering Research Council (NSERC)

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.

The calcium paradox - what should we have to fear?

The calcium paradox was first mentioned in 1966 by Zimmerman et al. Thereafter gained great interest from the scientific community due to the fact of the absence of calcium ions in heart muscle cells produce damage similar to ischemia-reperfusion. Although not all known mechanisms involved in cellular injury in the calcium paradox intercellular connection maintained only by nexus seems to have a key role in cellular fragmentation. The addition of small concentrations of calcium, calcium channel blockers, and hyponatraemia hypothermia are important to prevent any cellular damage during reperfusion solutions with physiological concentration of calcium.

Effects of Chronic Myocardial Infarction on Cardiac Muscle Performance and Structure In-Vivo and In-Vitro

After chronic myocardial infarction (CMI) the myocardium undergoes remodeling which is described initially as ‘hypertrophic’ followed by ‘de-compensation’. We have studied this phenomenon at the sarcomeric level to understand how muscle mechanics, post-translational modifications, tissue morphology and sarcomeric structures are affected in the remaining well-perfused tissue.We use a rat model of left anterior descending (LAD) ligation to create phenotypic chronic myocardial infarction (CMI). A specialized temperature jump (T-jump) approach assess changes to myocardial muscle function during physiological shortening in saturating (32μM) and physiological (1μM) calcium concentrations. During ‘compensation’ we observe higher power (45%) compared to control. During ‘de-compensation’ we observe a 50% reduction in peak power and 40% reduction in maximal unloaded shortening velocity (Vmax). We exploit second harmonic generation (SHG) by two-photon excitation microscopy (TPE) to assess collagen abundance and infiltration. Global type I collagen abundance and infiltration increases 2-fold post-AMI but does not change during disease progression. Phos-Tag SDS-PAGE is used to assess Troponin I (TnI), Myosin Binding Protein C (MyBP-C) and Regulatory Light Chain (RLC) phosphorylation. Electron microscopy is used to assess sarcomeric organization and mitochondrial health and abundance. Sarcomeric disorder becomes evident with disease progression as well as a global reduction in functional mitochondria. This study demonstrates that sarcomeric protein modification is an important modifier of cardiac muscle function during the evolution of disease, which mirrors the route of progression from ‘compensated’ to ‘decompensated’ phenotypes that is part of the wider picture of heart failure that encompasses changes to tissue structure and organelle morphology.

Ca2 + induces PI(4,5)P2 clusters on lipid bilayers at physiological PI(4,5)P2 and Ca2 + concentrations

Calcium has been shown to induce clustering of PI(4,5)P2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P2 (TopFluor(TF)-PI(4,5)P2), we show that Ca2+ promotes PI(4,5)P2 clustering in lipid bilayers at physiological concentrations of both Ca2+ and PI(4,5)P2. Fluorescence depolarization data of TF-PI(4,5)P2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P2 and calcium, the average cluster size comprises ~15 PI(4,5)P2 molecules. The presence of Ca2+-induced PI(4,5)P2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P2 clustering was more pronounced in liquid ordered (lo) membranes, and the PI(4,5)P2-Ca2+ clusters presented an increased affinity for lo domains. In this way, PI(4,5)P2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P2 clustering on lo domains might provide targeted nucleation sites for PI(4,5)P2 clusters upon calcium stimulus.

Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity

TG2 (transglutaminase 2) is a calcium-dependent protein cross-linking enzyme which is involved in a variety of cellular processes. The threshold level of calcium needed for endogenous and recombinant TG2 activity has been controversial, the former being more sensitive to calcium than the latter. In the present study we address this question by identifying a single amino acid change from conserved valine to glycine at position 224 in recombinant TG2 compared with the endogenous sequence present in the available genomic databases. Substituting a valine residue for Gly224 in the recombinant TG2 increased its calcium-binding affinity and transamidation activity 10-fold and isopeptidase activity severalfold, explaining the inactivity of widely used recombinant TG2 at physiological calcium concentrations. ITC (isothermal titration calorimetry) measurements showed 7-fold higher calcium-binding affinities for TG2 valine residues which could be activated inside cells. The two forms had comparable substrate- and GTP-binding affinities and also bound fibronectin similarly, but coeliac antibodies had a higher affinity for TG2 valine residues. Structural analysis indicated a higher stability for TG2 valine residues and a decrease in flexibility of the calcium-binding loop resulting in improved metal-binding affinity. The results of the present study suggest that Val224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells.

Treatment of cyathostominosis with ivermectin and its influence on selected blood biochemical parameters

Infections caused by nematodes of the subfamily Cyathostominae affect nearly 100% of pastured horses. Despite of an absence of pronounced symptoms, cyathostominosis can have very serious health consequences. The aim of this study was to monitor changes in total protein levels and concentrations of selected microelements and macroelements in the blood of horses before and after ivermectin treatment. In healthy horses infected by the studied parasites, total blood protein levels were below the physiological norm, but iron (Fe), magnesium (Mg), calcium (Ca) and phosphorus (P) concentrations were within normal limits. Ivermectin treatment reduced the number of excreted parasite eggs (FEC) by 100%, and dead parasites were observed in feces. Decreased iron (Fe) concentrations and an insignificant increase in total blood protein levels were reported. A progressive decline in iron levels was observed when parasite eggs reappeared in feces 60 days after treatment. Iron loss takes place as a result of bleeding from the large intestine when adult nematodes affected by the drug are removed from intestine and fourth-stage larvae leave parasitic nodules in the intestinal wall. A drop in iron levels could be an indirect indicator of the severity of cyathostominosis.

Increase of TNFα-stimulated Osteoarthritic Chondrocytes Apoptosis and Decrease of Matrix Metalloproteinases 9 by NF-κB Inhibition

ObjectiveTo investigate the in vitro effect of caffeic acid phenethyl ester (CAPE), a NF-κB inhibitor, on the apoptosis of osteoarthritic (OA) chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9). MethodsAnnexin V-FITC/propidium iodide (PI) labeling and western blotting were used to observe and determine the apoptosis in TNFα-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. ResultsIt was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFα for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated (from proMMP-9 to active MMP-9), and the physiologic calcium concentration (2.5 mmol/L) could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFα for 24 h. The stimulatory effect of TNFα just on proMMP-9 was counteracted significantly by CAPE. ConclusionNF-κB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFα (a pro-apoptotic factor). Therefore, therapeutic NF-κB inhibitor was a ‘double-edged swords’ to the apoptosis of chondrocytes and the secretion of MMP-9.

The PsbP family of proteins

The PsbP family of proteins consists of 11 evolutionarily related thylakoid lumenal components. These include the archetypal PsbP protein, which is an extrinsic subunit of eukaryotic photosystem II, three PsbP-like proteins (CyanoP of the prokaryotic cyanobacteria and green oxyphotobacteria, and the PPL1 and PPL2 proteins found in many eukaryotes), and seven PsbP-domain (PPD) proteins (PPD1-PPD7, most of which are found in the green plant lineage). All of these possess significant sequence and structural homologies while having very diverse functions. While the PsbP protein has been extensively studied and plays a functional role in the optimization of photosynthetic oxygen evolution at physiological calcium and chloride concentrations, the molecular functions of the other family members are poorly understood. Recent investigations have begun to illuminate the roles that these proteins play in membrane protein complex assembly/stability, hormone biosynthesis, and other metabolic processes. In this review we have examined this functional information within the context of recent advances examining the structure of these components.

Air–liquid and liquid–liquid interfaces influence the formation of the urothelial permeability barrier in vitro

Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air-liquid (AL) and liquid-liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.

Physiological Calcium Concentrations Induce PI(4,5)P2 Clustering: PI(4,5)P2 as a Lipidic Calcium Sensor

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the plasma membrane of eukaryotic cells that is essential for several cellular mechanisms, including organization and remodeling of the actin cytoskeleton, membrane trafficking, endocytosis and exocytosis. The regulation of these processes is thought to involve localized enrichment of PI(4,5)P2 in the plasma membrane at particular timings. Particularly in calcium-triggered SNARE-dependent exocytosis, PI(4,5)P2 is known to interact and co-segregate with certain synaptic proteins. However, the mechanisms responsible for PI(4,5)P2 clustering in particular sites of the plasma membrane are still not completely understood.Using membrane model systems and fluorescently labeled PI(4,5)P2, we were able to demonstrate that PI(4,5)P2 lateral distribution is sensitive to physiologic calcium concentrations, in the low micromolar range. Calcium concentrations of 0-100 μM were used to mimic neuron intracellular calcium concentrations before and after a stimulus. Fluorescence self-quenching and energy migration data obtained from steady-state and time-resolved fluorescence methods clearly showed that Ca2+ ions promoted the clustering of PI(4,5)P2 molecules in both liquid disordered and liquid ordered membranes. Sensitivity of PI(4,5)P2 clustering to [Ca2+] is shown to be highly dependent on both PI(4,5)P2 density and membrane lipid phase.In this way, the lateral distribution of PI(4,5)P2 presents significant sensitivity to changes in calcium concentration of only a few micromolar, allowing this lipid to function as a lipidic calcium sensor. In the particular case of SNARE-dependent exocytosis, it is expected that localized increases in calcium concentration will dramatically change the pattern of PI(4,5)P2 interaction with synaptic proteins, possibly enhancing fusion rates and promoting exocytosis.Acknowledgements: FCT Portugal is acknowledged for financial support (PTDC/QUI-BIQ/112067/2009; PTDC/QUI-BIQ/099947/2008; SFRH/BPD/64320/2009; SFRH/BD/80575/2011).

Imaging analysis of early DMP1 mediated dentine remineralization

ObjectiveThis study assessed the micro-morphological changes in demineralized dentine scaffold following incubation with recombinant dentine matrix protein 1 (rDMP1). DesignExtracted human molar crowns were sectioned into 6 beams (dimensions: 0.50mm×1.70mm×6.00mm), demineralized and incubated overnight in 3 different media (n=4): rDMP1 in bovine serum albumin (BSA), BSA and distilled water. Samples were placed in a chamber with simulated physiological concentrations of calcium and phosphate ions at constant pH 7.4. Samples were immediately processed for transmission electron microscopy (TEM) and field emission-scanning electron microscopy (FE-SEM) after 1 and 2 weeks. ResultsAnalysis of the scaffold showed that decalcification process retained the majority of endogenous proteoglycans and phosphoproteins. rDMP1 treated samples promoted deposition of amorphous calcium phosphate (ACP) precursors and needle shaped hydroxyapatite crystals surrounding collagen fibrils. The BSA group presented ACP bound to collagen with no needle-like apatite crystals. Samples kept in distilled water showed no evidence of ACP and crystal apatite. Results from rDMP1 immobilized on dentine matrix suggests that the acidic protein was able to bind to collagen fibrils and control formation of amorphous calcium phosphate and its subsequent transformation into hydroxyapatite crystals after 2 weeks. ConclusionThese findings suggest a possible bio-inspired strategy to promote remineralization of dentine for reparative and regenerative purposes.

Corrigendum to: Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

Corrigendum to: Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

Adaptation of Granule Cell to Purkinje Cell Synapses to High-Frequency Transmission

The mossy fiber (MF)-granule cell (GC) pathway conveys multiple modalities of information to the cerebellar cortex, converging on Purkinje cells (PC), the sole output of the cerebellar cortex. Recent in vivo experiments have shown that activity in GCs varies from tonic firing at a few hertz to phasic bursts >500 Hz. However, the responses of parallel fiber (PF)-PC synapses to this wide range of input frequencies are unknown, and there is controversy regarding several frequency-related parameters of transmission at this synapse. We performed recordings of unitary synapses and combined variance-mean analysis with a carefully adapted extracellular stimulation method in young and adult rats. We show that, although the probability of release at individual sites is low at physiological calcium concentration, PF-PC synapses release one or more vesicles with a probability of 0.44 at 1.5 mm [Ca(2+)](e). Paired-pulse facilitation was observed over a wide range of frequencies; it renders burst inputs particularly effective and reproducible. These properties are primarily independent of synaptic weight and age. Furthermore, we show that the PF-PC synapse is able to sustain transmission at very high frequencies for tens of stimuli, as a result of accelerated vesicle replenishment and an apparent recruitment of release site vesicles, which appears to be a central mechanism of paired-pulse facilitation at this synapse. These properties ensure that PF-PC synapses possess a dynamic range enabling the temporal code of MF inputs to be transmitted reliably to the PC.

Calcium stabilizes the von Willebrand factor A2 domain by promoting refolding

Von Willebrand factor (VWF) is a large, multimeric plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Very large VWF multimers have the greatest thrombogenic activity, which is attenuated by cleavage in the A2 domain by the metalloproteinase ADAMTS13. ADAMTS13 proteolysis requires mechanical force to expose the scissile bond and is regulated by a calcium-binding site within A2. In this study, we characterized the interaction between VWF A2 and calcium by examining the effect of calcium on VWF A2 stability and mechanical unfolding and refolding. Isothermal calorimetry yielded a calcium binding K(d)=3.8±1.0μM and reversible thermal denaturation showed that 5mM calcium stabilized the unfolding transition from 56.7±0.1 to 69.1±0.1°C. Using optical tweezers to apply tensile force to single domains, we found that calcium did not affect VWF A2 unfolding, but rather enhanced refolding kinetics fivefold, resulting in a 0.9kcal/mol stabilization in the folding activation energy in the presence of calcium. Taken together, our data demonstrate that VWF binds calcium at physiologic calcium concentrations and that calcium stabilizes VWF A2 by accelerating refolding.

Pushing the Envelope of Sound

Normal hearing depends on the amplification of sound in the cochlea. In this issue of Neuron, Fettiplace and colleagues show that prestin-based amplification is a viable mechanism as it is not limited by the outer hair cell's membrane time constant as previously surmised.

Prestin-Driven Cochlear Amplification Is Not Limited by the Outer Hair Cell Membrane Time Constant

SummaryOuter hair cells (OHCs) provide amplification in the mammalian cochlea using somatic force generation underpinned by voltage-dependent conformational changes of the motor protein prestin. However, prestin must be gated by changes in membrane potential on a cycle-by-cycle basis and the periodic component of the receptor potential may be greatly attenuated by low-pass filtering due to the OHC time constant (τm), questioning the functional relevance of this mechanism. Here, we measured τm from OHCs with a range of characteristic frequencies (CF) and found that, at physiological endolymphatic calcium concentrations, approximately half of the mechanotransducer (MT) channels are opened at rest, depolarizing the membrane potential to near −40 mV. The depolarized resting potential activates a voltage-dependent K+ conductance, thus minimizing τm and expanding the membrane filter so there is little receptor potential attenuation at the cell's CF. These data suggest that minimal τm filtering in vivo ensures optimal activation of prestin.

Abstract 3126: Intercellular communication in the urothelium: Evidence for a switch in gap junction regulation during differentiation

Abstract Cells have a requirement for intercellular communication in order to maintain normal tissue polarity and homeostasis. Knowledge of the signaling molecules transferred through specific gap junctions is limited; however aberrant expression of connexins, the protein subunits of gap junctions, has been linked to many disorders including neoplastic transformation of brain and colorectal tissues. A number of reports have documented changes in connexin expression associated with bladder cancer. The aims of this study were 1) to define the connexins expressed by the epithelial lining of the human urinary tract (urothelium) in situ and in vitro; 2) to determine if expressed connexins assemble into functional gap junctions and 3) the contribution of specific connexins to urothelial tissue physiology. Established culture systems for normal human urothelial (NHU) cells were used to replicate proliferative (regenerative) or differentiated cell phenotypes. RT-PCR was used to screen normal urothelial tissue and NHU cell cultures for expression of connexin genes. Immunochemical techniques were employed to determine connexin expression and localization in situ and in vitro. Dye-transfer techniques were used on cultured cells to confirm that assembled gap junctions supported functional cell-cell communication. Of the 20 human connexin genes, six were expressed in situ and by differentiated cultures, whilst two were expressed only in proliferative culture conditions. NHU cells were capable of dye transfer in both proliferative and differentiated cultures, indicating the presence of functional gap junctions. Immunofluorescence microscopy demonstrated that the switch in medium from a low (0.09mM) to near physiological (2mM) calcium concentration resulted in increased assembly of gap junctions containing connexin-43. Connexin-32 was only expressed by differentiated cultures, as shown by RT-PCR and immunochemistry, where it was localized to intercellular borders indicating a functional role. This data represents the first systematic characterization of connexin family expression in the urothelium and provides evidence that urothelial cells alter their connexin expression upon differentiation to produce specific channels. Connexin-32 has been reported to be involved in terminal differentiation of human mammary gland and future work will focus on characterizing the role of connexins in defining the differentiated urothelial phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2011-3126