Characterization of a Membrane Calcium Pathway Induced by Rotavirus Infection in Cultured Cells

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na⁺, K⁺, and Ca²⁺ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca²⁺ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca²⁺ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca²⁺, Ba²⁺, Sr²⁺, Mn²⁺, and Co²⁺. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La³⁺ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca²⁺ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca²⁺ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca²⁺ pools required for virus maturation and cell death.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Regulation of the Fast Vacuolar Channel by Cytosolic and Vacuolar Potassium

At resting cytosolic Ca²⁺, passive K⁺ conductance of a higher plant tonoplast is likely dominated by fast vacuolar (FV) channels. This patch-clamp study describes K⁺-sensing behavior of FV channels in Beta vulgaris taproot vacuoles. Variation of K⁺ between 10 and 400 mM had little effect on the FV channel conductance, but a pronounced one on the open probability. Shift of the voltage dependence by cytosolic K⁺ could be explained by screening of the negative surface charge with a density σ = 0.25 e⁻/nm². Vacuolar K⁺ had a specific effect on the FV channel gating at negative potentials without significant effect on closed-open transitions at positive ones. Due to K⁺ effects at either membrane side, the potential at which the FV channel has minimal activity was always situated at ~50 mV below the potassium equilibrium potential, E_K⁺. At tonoplast potentials below or equal to E_K⁺, the FV channel open probability was almost independent on the cytosolic K⁺ but varied in a proportion to the vacuolar K⁺. Therefore, the release of K⁺ from the vacuole via FV channels could be controlled by the vacuolar K⁺ in a feedback manner; the more K⁺ is lost the lower will be the transport rate.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell''s diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.

Methodology/Principal Findings

The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.

Conclusions/Significance

The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.     

Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA

In the present study we describe the analysis of optically recorded whole cell Ca²⁺ transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca²⁺ mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca²⁺-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca²⁺ to the myoplasmic space (Ca²⁺ input flux). The validity of the procedure was confirmed by model simulations using artificial Ca²⁺ input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca²⁺ input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca²⁺ flux carried by the L-type Ca²⁺ channels, indicating that it consists mainly of voltage-controlled Ca²⁺ release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca²⁺ inward current, indicating that Ca²⁺ release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca²⁺ release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.     

Rat Merkel Cells Are Mechanoreceptors and Osmoreceptors

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca²⁺ channels and hypotonic-induced membrane deformation is known to lead to Ca²⁺ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification. Their properties were investigated through electrophysiological studies. Voltage-gated K⁺, Ca²⁺ and Ca²⁺-activated K⁺ (K_Ca) channels were identified, as previously described. Here, we also report the activation of Ca²⁺ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca²⁺ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca²⁺ chelator EGTA. Endosome fusion was restored by the addition of Ca²⁺ with an optimum at a free Ca²⁺ concentration of 0.3 × 10⁻⁶ M. Other divalent cations did not substitute for Ca²⁺. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca²⁺. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca²⁺ from the endosomal interior.     

Light-induced changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H⁺, Ca²⁺, K⁺, and Cl⁻ fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia faba L.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca²⁺ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H⁺, K⁺, and Cl⁻ occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca²⁺ began within the time resolution of our measurements (5 s). In the absence of H⁺ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO₂ uptake by photosynthesizing tissue, whereas activation of the plasma membrane H⁺ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO₂ diffusion into the leaf.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes

Mitochondria act as potent buffers of intracellular Ca²⁺ in many cells, but a more active role in modulating the generation of Ca²⁺ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca²⁺ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca²⁺ store with thapsigargin (TG) activates Ca²⁺ release-activated Ca²⁺ (CRAC) channels in T cells, and the ensuing influx of Ca²⁺ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca²⁺ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na⁺ depletion, which inhibits Na⁺-dependent Ca²⁺ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca²⁺ levels that are consistent with its localization in the TG-insensitive store. Ca²⁺ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca²⁺), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca²⁺ uptake is sensitive to extracellular [Ca²⁺], indicating that mitochondria sense Ca²⁺ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na⁺ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca²⁺ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca²⁺ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca²⁺ that enters T cells via store-operated Ca²⁺ channels, but also play an active role in modulating the rate of capacitative Ca²⁺ entry.     

Alkali Metal Cation Transport and Homeostasis in Yeasts

Summary: The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K⁺ and Na⁺, is of utmost importance for living cells, since they determine cell volume, intracellular pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K⁺ transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na⁺ can be tolerated due to the existence of an Na⁺, K⁺-ATPase and an Na⁺, K⁺/H⁺-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.     

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca²⁺-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca²⁺ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca²⁺ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca²⁺ is quenched to or below resting intracellular Ca²⁺ concentration ([Ca²⁺]_e [Ca²⁺]_i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca²⁺]_e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca²⁺]_i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca²⁺ mobilization from stores (providing close to threshold Ca²⁺ levels) and Ca²⁺ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca²⁺ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.