Vacuolar H+/Ca2+ transport: who's directing the traffic?

Physiological studies have established the role of plant high-capacity vacuolar H+/Ca2+ exchange activity in ion homeostasis and signal transduction. The molecular characterization and structure-function analyses of these transporters are just beginning to emerge. In yeast, Ca2+ signaling molecules regulate vacuolar H+/Ca2+ exchange. Recently, some of the Ca2+ dependent "molecular relay" molecules have been characterized in plants; however, the regulation of plant vacuolar H+/Ca2+ exchange remains an open question.     

Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum?

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium-free medium containing magnesium and calcium, at pH 6 and 20°C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane-bound ionophore A23187, the observed turnoverdependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

How Does Regulatory Ca2+ Regulate the Na+-Ca2+ Exchanger?

Spatial and temporal regulation of intracellular Ca²⁺ concentrations is a fundamental requirement for life. The mammalian cardiac Na⁺-Ca²⁺ exchanger serves as the main mechanism for Ca²⁺ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca²⁺, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca²⁺. Together with mutational analysis of the Ca²⁺ binding sites, this study reveals the crucial role of one of the two bound Ca²⁺ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Aggregation and collapse of fungal wall vesicles in hyphal tips: a model for the origin of the Spitzenk?rper

The intracellular origins of polarity and branch initiation in fungi centre upon a localization in the supply of fungal wall constituents to specific regions on the hyphal wall. Polarity is achieved and maintained by accumulating secretory vesicles, prior to incorporation into the wall, in the form of an apical body or Spitzenkörper. However, neither the mechanisms leading to this accumulation nor the initiation of branching, are as yet understood. We propose a mechanism, based on experimental evidence, which considers the mechanical properties of the cytoskeleton in order to explain these phenomena. Cytoskeletal viscoelastic forces are hypothesized to be responsible for biasing vesicles in their motion, and a mathematical model is derived to take these considerations into account. We find that, as a natural consequence of the assumed interactions between vesicles and cytoskeleton, wall vesicles aggregate in a localized region close to the tip apex. These results are used to interpret the origin of the Spitzenkörper. The model also shows that an aggregation peak can collapse and give rise to two new centres of aggregation coexisting near the tip. We interpret this as a mechanism for apical branching, in agreement with published observations. We also investigate the consequences and presumptive role of vesicle–cytoskeleton interactions in the migration of satellite Spitzenkörper. The results of this work strongly suggest that the formation of the Spitzenkörper and the series of dynamical events leading to hyphal branching arise as a consequence of the bias in vesicle motion resulting from interactions with the cytoskeleton.     

Is all of the endoplasmic reticulum created equal? The effects of the heterogeneous distribution of endoplasmic reticulum Ca2+-handling proteins

The endoplasmic reticulum is a heterogeneous compartment with respect to the distribution of its Ca2+-handling proteins, namely the Ca2+-binding proteins, the Ca2+ pumps and the Ca2+ release channels. The nonuniform distribution of these proteins may explain the functional heterogeneity of the endoplasmic reticulum, such as the generation of spatially complex Ca2+ signals, Ca2+ homeostasis, and protein folding and quality control.     

An additional step in the transmission of Yersinia pestis?

Plague, caused by the bacterium Yersinia pestis, is a mammalian vector-borne disease, transmitted by fleas that serve as the vector between rodent hosts. For many pathogens, including Y. pestis, there are strong evolutionary pressures that lead to a reduction in ‘useless genes'', with only those retained that reflect function in the specific environment inhabited by the pathogen. Genetic traits critical for survival and transmission between two environments, the rodent and the flea, are conserved in epizootic/epidemic plague strains. However, there are genes that remain conserved for which no function in the flea–rodent cycle has yet been observed, indicating an additional environment may exist in the transmission cycle of plague. Here, we present evidence for highly conserved genes that suggests a role in the persistence of Y. pestis after death of its host. Furthermore, maintenance of these genes points to Y. pestis traversing a post-mortem path between, and possibly within, epizootic periods and offering insight into mechanisms that may allow Y. pestis an alternative route of transmission in the natural environment.     

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca²⁺ in the Ca²⁺-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca²⁺-uptake, and heat up during ATP synthesis and Ca²⁺-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca²⁺-ATPases, not only act as Ca²⁺-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

An investigation of the effects of Ca²+ channel inhibitors on branching and chemotropism in the oomycete Achlya bisexualis: Support for a role for Ca²+ in apical dominance

In an attempt to better understand branching and chemotropism, we describe the effects of Ca2+ channel inhibitors on these processes in Achlya bisexualis, using a branch induction technique and whole plate assays. Branching appears to be a two step process with the initial formation of a bump from which a branch emerges. Verapamil increased numbers of branches in whole plate assays and decreased the distance from the first branch to the tip. In induction assays verapamil increased the number of bumps formed, although in some hyphae it inhibited the transition from an initial bump to a branch. When a branch formed it did not affect the time taken to branch. It had no effect on chemotropism. Lanthanum (La3+) and gadolinium (Gd3+) also increased branching in whole plate assays but their effect was much less marked and they had no effect on bump/branch number in induction assays. Gd3+ decreased the time taken to branch. Both La3+ and Gd3+ increased chemotropism. These data suggest firstly that the respective inhibitors may affect different parts of the branching process and secondly that Ca2+ influx through channels may not be a requirement for branching, indeed such movements may suppress branching. This would fit with elevated Ca2+ at the tip playing a role in apical dominance.     

Possible role for Ca2+ in the pathophysiology of the prion protein?

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease.     

Can a Ca2+ pump in the endoplasmic reticulum of the Lepidium root be the trigger for rapid changes in membrane potential after gravistimulation?

Since gravistimulation is followed by alterations in the external current symmetry (Behrens et al., 1982), the effect of gravistimulation on cellular membrane potential was investigated using conventional glass microelectrode techniques. The resting potential of statocytes in a vertically oriented root is approx. -118 mV. Upon gravistimulation, the membrane potential is temporarily depolarized (lag time = 2 s) to a potential of approx. -93 mV. This depolarization is only observed in statocytes located on the physically lower root flank while those on the corresponding upper flank become weakly hyperpolarized (approx. -13 mV). These results reflect altered ion fluxes across the plasma membrane. The perception of gravistimulus was suggested to result from a pressure of the amyloplasts on the distal endoplasmic reticulum (ER) of the statocytes (Sievers and Volkmann, 1972). A causal relationship between changes in ER-amyloplast interactions and the rapid alterations in plasma membrane potential described above is not known. A candidate for such an intracellular messenger is Ca2+. As a first step in establishing the validity of such an assumption, we have isolated ER membranes from roots. When incubated with micromolar concentrations of Ca2+, the vesicular membrane fraction accumulates Ca2+. The accumulation is ATP-dependent and -specific and is directly coupled to ATP hydrolysis since a protonophore shows no inhibitory effect. Thus, in analogy to the sarcoplasmic reticulum of muscle, regulation of an ER-localized Ca2+ compartment might be an important step in such complex processes as stimulus-transduction in gravitropism.     

Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy

Pig coronary artery smooth muscle expresses the Na⁺–Ca²⁺-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca²⁺ pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca²⁺ pool. Caveolae may also direct Ca²⁺ traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca²⁺ for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca²⁺ signaling pathways but not others.     

Lipids in Ca2+ signalling—An introduction

Lipids and lipid-derived metabolites are increasingly recognised as bona fide signalling molecules that regulate many cellular processes. These include the well-established InsP₃, diacylglycerol (DAG), PIP₂, PIP₃ and arachidonic acid (AA), as well as other poly-unsaturated fatty acids (PUFAs), lysophospholipids, sphingolipids, endocannabinoids and endovanilloids. They regulate a plethora of molecules that are involved in Ca²⁺ signalling, including various ion channels, pumps and transporters, thereby triggering, modulating and fine-tuning Ca²⁺ signals. Although appreciated individually, it seems timely to highlight the overall impact of lipids as signalling molecules and their role in Ca²⁺ signalling, and this is the aim of this special issue of Cell Calcium.     

Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background

Ca²⁺ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) in the prefusion phase, the occurrence and significance of Ca²⁺ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca²⁺- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca²⁺]_c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t_1/2 of decay = 3.2 s) rise of localized [Ca²⁺]_c originating at the site of lamellar body fusion. [Ca²⁺]_c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca²⁺ from, or addition of Ni²⁺ to the extracellular solution, strongly inhibited these [Ca²⁺]_c transients, whereas Ca²⁺ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca²⁺]_c. Both effects were reduced by the non-specific Ca²⁺ channel blocker {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365.

Conclusions/Significance

Fusion-activated Ca²⁺ entry (FACE) is a new mechanism that leads to [Ca²⁺]_c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca²⁺ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.     

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Smoke-derived cues in the regulation of seed germination: are Ca2+-dependent signals involved?

Plant Growth Regulation - Seed dormancy and germination are two distinct physiological processes in the life cycle of plants. Dormancy alleviation and the attendant transition to seed germination...