Low-affinity Ca2 Research Articles

Metal binding study of calreticulin: An immunomodulatory protein of human filarial parasite Brugia malayi

Calreticulin (CRT), a highly conserved ubiquitous eukaryotic protein with a molecular mass of 46 kDa, containing three domains (N, P, and C) is involved in promoting prolonged parasite-host relations. Brugia malayi Calreticulin (BmCRT) is involved in the establishment of parasite infection by suppression of C1q-mediated host immune response. Calcium plays important role in this immunomodulatory mechanism of BmCRT. In the present study binding of calcium with BmCRT region involved in this interaction was investigated and correlated with the accompanying changes in fluorescence, circular dichroism (CD) and UV absorption. The results obtained clearly indicated that BmCRT is a calcium binding protein and contains types two of Ca2+ binding sites, one high affinity Ca2+ binding site at P domain and another low affinity Ca2+ binding site at C domain. Zinc also binds to additional sites that do not have appreciable affinity for the calcium. These studies have provided new knowledge that allows us to describe how the structure of BmCRT responds to interactions with calcium and zinc which is different from human CRT and also discuss how this mechanism help to complex formation with host C1q.

Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform.

The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca2+ channels and genetically encoded Ca2+ indicator (GECI). We observed that targeting GECI to the plasma membrane was essential for efficient loading GECI in the VLPs. Although the physiological Ca2+ concentration [Ca2+] maintained in the cells was low (∼10 nM), the concentration in the VLPs was high. This prevented the detection of the increase in [Ca2+] caused by binding of the ligand to the receptor. To address this problem, we employed Lyn-R-CEPIA1, which had low affinity for Ca2+, and a membrane targeting sequence. Thus, we succeeded in monitoring the activation of cyclic nucleotide-gated channels (CNG) on the VLPs by measuring the increase in fluorescence of Lyn-R-CEPIA1. Our VLP-based sensing system can act as a fundamental platform for all kinds of ligand-gated channels.

Roles of three Fusarium graminearum membrane Ca2+ channels in the formation of Ca2+ signatures, growth, development, pathogenicity and mycotoxin production

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca2+ in fungi. Such changes are referred as the Ca2+ signature and control cellular responses by modulating the activity or location of diverse Ca2+-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca2+ signaling, therefore, characterization of how Ca2+ moves to and from the cytoplasm to create Ca2+ signatures under different conditions is fundamental. Three genes encoding plasma membrane Ca2+ channels in a Fusarium graminearum strain that expresses a fluorescent protein-based Ca2+ indicator in the cytoplasm were mutagenized to investigate their roles in the generation of Ca2+ signatures under different growth conditions and genetic backgrounds. The genes disrupted include CCH1 and MID1, which encode a high affinity Ca2+ uptake system, and FIG1, encoding a low affinity Ca2+ channel. Resulting mutants were also analyzed for growth, development, pathogenicity and mycotoxin production to determine how loss of each of the genes alters these traits. To investigate whether individual genes influence the function and expression of other genes, phenotypes and Ca2+ signatures of their double and triple mutants, as well as their expression patterns, were analyzed.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100μm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD =543±51nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38±0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.

Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically encoded ER-targeted low-affinity Ca2+ indicators optimized for examining axonal ER Ca2+. Our experiments revealed that presynaptic function is tightly controlled by ER Ca2+ content. We found that neuronal activity drives net Ca2+ uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca2+ except during prolonged repetitive firing. In contrast, we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca2+]ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca2+ entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca2 + concentrations

Ca2+ signaling is a major signal transduction pathway involved in T cell activation, but also in apoptosis of T cells. Since T cells make use of several Ca2+-mobilizing second messengers, such as nicotinic acid adenine dinucleotide phosphate, d-myo-inositol 1,4,5-trisphosphate, and cyclic ADP-ribose, we intended to analyze luminal Ca2+ concentration upon cell activation. Mag-Fluo4/AM, a low-affinity Ca2+ dye known to localize to the endoplasmic reticular lumen in many cell types, showed superior brightness and bleaching stability, but, surprisingly, co-localized with mito-tracker, but not with ER-tracker in Jurkat T cells. Thus, we used Mag-Fluo4/AM to monitor the free luminal mitochondrial Ca2+ concentration ([Ca2+]mito) in these cells. Simultaneous analysis of the free cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]mito upon cell stimulation revealed that Ca2+ signals in the majority of mitochondria were initiated at [Ca2+ ]i≥approx. 400 to 550nM.In primary murine CD4+ T cells, Mag-Fluo4 showed two different localization patterns: either co-localization with mito-tracker, as in Jurkat T cells, or with ER-tracker. Thus, in single primary murine CD4+ T cells, either decreases of [Ca2+ ]ER or increases of [Ca2+ ]mito were observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The osteoclasts attach to the bone surface where the extracellular calcium concentration decreases.

Osteoclasts demineralize and resorb bone once they attach to its surface. However, it's still unclear how the osteoclasts choose the specific sites for their attachments. It is postulated in this article that the decreased extracellular free ionized calcium concentration (Ca2+[e]) can provide a microenvironment for osteoclasts to recognize and then initiate the attachment process. The osteoclasts initially attach to the bone surface via integrating its integrin αvβ3 and RGD containing ligands in bone matrix. Through the interaction with RGD-containing ligand, the integrin αvβ3 forms carboxylate oxygen noncovalent, which is further stabilized by accompanied electrostatic interaction between the Ca2+ and the β3 subunit. There are two types of cation-binding sites on the β3 subunit: the high affinity Ca2+ binding site ("LC" site) that activates the osteoclasts by promoting the combination; the low affinity Ca2+ binding site ("I" site, also named ADMIDAS) that deactivates the osteoclasts by dissociating the combination and it can override the "LC" site's positive effect on osteoclasts when necessary. Normally the Ca2+ concentration of bone extracellular fluid is maintained within a normal range by osteocytes, keeping the "I" sites activated. When the osteocytes' function stalls and the ambient Ca2+ concentration falls below the affinity discrimination threshold between the "I" site and the "LC" site, the "LC" site's promoting function starts to out compete the "I" site in its inhibitory effect, in which case the inactive integrin αvβ3 turns into an extended active form and the osteoclasts start to attach, signifying the initiation of bone resorption.

Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER.

Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5.

Real-time intra-store confocal Ca2+ imaging in isolated mouse cardiomyocytes

To initiate the contraction of cardiomyocytes, Ca2+ is released from the SR to the cytosol via ryanodine receptors (RyRs), which are activated by the Ca2+–induced Ca2+ release mechanism (CICR). The activity of RyRs is regulated by both, cytosolic and SR luminal Ca2+. Deregulation of the CICR, by dysfunctional SR Ca2+ release or uptake, is frequently associated with cardiac pathologies (e.g. arrhythmias, CPVT, heart failure). Recently, the interest to directly measure changes of the free Ca2+ concentration within the SR ([Ca2+]SR) has led to the application of low affinity Ca2+ indicators (mag-fluo-4, Fluo-5N) to follow changes of [Ca2+]SR in cardiomyocytes from some species. However, direct measurement of Ca2+ signals from the SR have not been possible in freshly isolated mouse cardiomyocytes. Here, we show a new protocol optimized to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol permits the design of experimental studies with direct evaluation of SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins. The technique, in combination with these models, will help to understand how these diseases and mutations affect Ca2+ signals within the SR and the Ca2+ sensitivity of the RyRs for cytosolic and SR luminal Ca2+, thereby contributing to arrhythmias or weak heart beat.

Can liming reduce barium uptake by agricultural plants grown on sandy soil?

AbstractUptake of barium (Ba) from soil by vascular plants varies among species. Despite the toxicity of soluble Ba compounds to plants, research on mechanisms controlling Ba uptake from natural soils is scarce. This study investigated the treatment effect of lime (CaCO3) added to a sandy soil containing a total of 500 mg Ba kg−1 on uptake and interspecies distribution of Ba in legumes and other cultivated food and feed plants. Nine species of grasses, vegetables, herbs, and legumes were cultivated under controlled conditions in a greenhouse experiment. The plants were harvested at maturity or flowering, dried, milled, and digested with nitric acid using the microwave technique prior to ICP‐MS analysis. All plant species acquired Ba from the soil in considerable amounts, probably due to low Ba adsorption potential of the sandy soil. Shoot tissue concentrations ranged from about 100 (grass) to 600 mg Ba kg−1 (legume) and root concentrations from about 100 (tuber vegetable) to 700 mg kg−1 (legume). Vicia cracca L. (bird vetch) showed an accumulation capacity due to high shoot concentrations of Ba compared to the other species. Higher yield accompanied by a potential dilution effect can partly explain why Trifolium repens L. (white clover), Pisum sativum ssp. arvénse L. (gray pea) and Hordeum vulgare L. (barley) did not display the highest Ba concentrations, but showed the highest Ba uptake from soil. High plant uptake of calcium (Ca) also seemed to enhance Ba uptake, by legumes in particular. However, liming reduced shoot Ba concentrations, particularly of species with low affinity for Ca. The risk of Ba accumulation thus raises toxicity concerns when forage legumes are cultivated in soils containing elevated concentrations of Ba.

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca2+ Dynamics in High-Ca2+ Organelles

Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe aratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) exvivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) invivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease.

R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes.

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR; [Ca]SR) partially determines the amplitude of cytosolic Ca transient that, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best approach available to directly measure [Ca]SR was to use low-affinity Ca indicators (e.g., Fluo-5N). However, this approach presents several limitations, including nonspecific cellular localization, dye extrusion, and species limitation. Recently a new genetically encoded family of Ca indicators has been generated, named Ca-measuring organelle-entrapped protein indicators (CEPIA). Here, we tested the red fluorescence SR-targeted Ca sensor (R-CEPIA1er) as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat ventricular myocytes with an adenovirus expressing the R-CEPIA1er gene displayed prominent localization in the SR and nuclear envelope. Calibration of R-CEPIA1er in myocytes resulted in a Kd of 609 μM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR [Ca]SR dynamics measured with R-CEPIA1er were compared with [Ca]SR measured with Fluo5-N. We found that both the time course of the [Ca]SR depletion and fractional SR Ca release induced by an action potential were similar between these two Ca sensors. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, measurement of [Ca]SR with R-CEPIA1er can be combined with cytosolic [Ca] measurements (with Fluo-4) to obtain more detailed information regarding Ca handling in cardiac myocytes. In conclusion, R-CEPIA1er is a promising tool that can be used to measure [Ca]SR dynamics in myocytes from different animal species.

Functional Coupling between TRPV1 and ANO1 in Sensory Neurons Requires Ca2+-Release from the Endoplasmic Reticulum

Ca2+ activated Cl- channel ANO1 (TMEM16A) is implicated in important physiological processes such as secretion, olfaction and inflammatory pain. Due to the low Ca2+ sensitivity, ANO1 channels require close association with the Ca2+ source to allow activation and preferential coupling of ANO1 to the endoplasmic reticulum (ER) in sensory neurons has been demonstrated. TRPV1 is another sensory ion channel implicated in inflammatory pain, which also activates ANO1. To investigate how TRPV1 Ca2+ signals may activate ANO1, we simultaneously monitored activity of ANO1 in dorsal root ganglion neurons using a halide-sensitive EYFP mutant (H148Q/I152L) and fura-2 Ca2+ imaging. Capsaicin application produced large Ca2+ transients with concurrent significant EYFP quenching (13.6±0.03%) compared to that induced by bradykinin (8.8±0.01%) and voltage-gated Ca2+ channel (VGCC) activation (6.02±0.006%). However, depleting intracellular Ca2+ stores with thapsigargin significantly reduced the Ca2+ rises and EYFP quenching. Interestingly, application of capsaicin in Ca2+-free extracellular solution did not abolish Ca2+ transients, which were significantly smaller (R/R0=1.6±0.17) compared to those produced by capsaicin in Ca2+-containing extracellular solutions (R/R0=2.6±0.29) or by bradykinin in Ca2+-free solutions (R/R0=2.0±0.31). Capsaicin applied in the Ca2+-free solution was unable to produce significant YFP quenching. Ca2+ imaging with the low affinity Ca2+ indicator fura-FF showed that capsaicin induced Ca2+ rises were significantly greater compared to bradykinin- or VGCC-induced rises. Finally, we demonstrated that the ER moved towards the plasma membrane after capsaicin application to TRPV1-overexpressing HEK cells, as revealed by TIRF microscopy. Taken together, these results indicate that for effective coupling of TRPV1 and ANO1 channels both Ca2+ influx and Ca2+ release from the ER are necessary. We further suggest that TRPV1 activation results in mobilisation of internal Ca2+ in which the ER may move closer to the membrane to facilitate ANO1 activation.

Strategic Composition and Enrichment of the Mitochondrial Ca2+ Uniporter at Mitochondria-SR Associations Creates Hotspots for Mitochondrial Ca2+ Uptake in the Cardiac Muscle

The bioenergetic fueling of cardiac muscle activity is tuned by Ca2+ signals propagated from SR to the mitochondrial matrix (excitation-bioenergetics coupling). Effective activation of the low-affinity mitochondrial Ca2+ uniporter (mtCU) is promoted by high [Ca2+] nanodomains where SR is tethered to mitochondria. Cardiac mitochondria have very large inner membrane (IMM) surface with extensive cristae folding but their mtCU current density is reportedly extremely low. We hypothesized that efficient excitation-bioenergetics coupling would require biased mtCU activity towards mitochondria-SR associations by mtCU being there more abundant and/or more efficient. To this end, we first analyzed the submitochondrial distribution of mtCU's pore protein, MCU in rat and mouse heart. Immunofluorescence (IF) in cells showed significant non-mitochondrial crossreactions; hence, we performed IF on mitochondria isolated from cardiac muscle (mtIF). mtIF showed 50% of punctate MCU positive structures colocalized with type-2 ryanodine receptor, suggesting a location bias to mitochondria-SR associations. Further, in submitochondrial membrane fractions MCU and the SR marker calsequestrin were both enriched with the inner-outer membrane contact points. Mitochondrial membranes in the SR fraction (MMSR) harbored as high concentrations of MCU as the crude mitochondria and much higher levels of the essential MCU regulator EMRE. Gel-filtration analysis showed size profiles of non-denaturing detergent-solubilized MCU- vs. EMRE-containing complexes largely overlapping in the SR fraction, while revealed MCU containing complexes without EMRE in the crude mitochondria. Finally, Ca2+ flux assays revealed more efficient mtCU-mediated Ca2+ uptake by the MMSR than the crude mitochondria. Our data thus suggest a distribution bias in the mtCU channel components over the cardiac IMM, turning mitochondria-SR interfaces to Ca2+ uptake ‘hotspots’ harboring all-EMRE-supported MCU while leaving other IMM areas less responsive to Ca2+ owing to EMRE-less mtCUs.

R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats).

A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast.

In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA (calcium cation exchangers) superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca2+ starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca2+ sensitivity of K667 (low affinity Ca2+ uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na+, Li+, Fe2+, Zn2+ and Co2+ and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K+ in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil.

Calsequestrins in skeletal and cardiac muscle from adult Danio rerio.

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.