KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

Loss of Pde6 reduces cell body Ca2+ transients within photoreceptors

Modulation of Ca²⁺ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca²⁺ indicators to define changes in perikaryal Ca²⁺ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca²⁺]_i levels within the cell body. Arguing against Ca²⁺ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca²⁺ fluxes revealed that degeneration of pde6c^w59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca²⁺]_i. Analysis of [Ca²⁺]_i in dissociated Pde6β^rd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca²⁺ elevations (‘flashes'') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca²⁺ fluxes diminished. This study thus provides insight into Ca²⁺ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca²⁺]_i that occurs in normal cones.     

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.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

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.     

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel

Increasing evidence suggests that changes in cytosolic Ca²⁺ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca²⁺ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca²⁺-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca²⁺ dependent. CDPK exhibits a Ca²⁺-induced electrophoretic mobility shift and its Ca²⁺-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca²⁺ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca²⁺-dependent protein phosphatase calcineurin enhances the Ca²⁺-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K⁺ channel KAT1 protein in a Ca²⁺-dependent manner. These results suggest that CDPK may be an important component of Ca²⁺ signaling in guard cells.     

Extramitochondrial Ca2+ in the Nanomolar Range Regulates Glutamate-Dependent Oxidative Phosphorylation on Demand

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca²⁺ in physiological concentration ranges (S_0.5 = 360 nM Ca²⁺). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca²⁺ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca²⁺-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca²⁺. At micromolar concentrations, Ca²⁺ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca²⁺ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca²⁺.     

Programmed Cellular Necrosis Mediated by the Pore-Forming α-Toxin from Clostridium septicum

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The α-toxin of Clostridium septicum is a β-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that α-toxin formed Ca²⁺-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca²⁺ levels. This Ca²⁺ influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca²⁺ influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of α-toxin-treated cells. Collectively, these data show that α-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced.     

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.     

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.     

Salinomycin induces calpain and cytochrome c-mediated neuronal cell death

Salinomycin is a polyether antibiotic with properties of an ionophore, which is commonly used as cocciodiostatic drug and has been shown to be highly effective in the elimination of cancer stem cells (CSCs) both in vitro and in vivo. One important caveat for the potential clinical application of salinomycin is its marked neural and muscular toxicity. In the present study we show that salinomycin in concentrations effective against CSCs exerts profound toxicity towards both dorsal root ganglia as well as Schwann cells. This toxic effect is mediated by elevated cytosolic Na⁺ concentrations, which in turn cause an increase of cytosolic Ca²⁺ by means of Na⁺/Ca²⁺ exchangers (NCXs) in the plasma membrane as well as the mitochondria. Elevated Ca²⁺ then leads to calpain activation, which triggers caspase-dependent apoptosis involving caspases 12, 9 and 3. In addition, cytochrome c released from depolarized mitochondria directly activates caspase 9. Combined inhibition of calpain and the mitochondrial NCXs resulted in significantly decreased cytotoxicity and was comparable to caspase 3 inhibition. These findings improve our understanding of mechanisms involved in the pathogenesis of peripheral neuropathy and are important to devise strategies for the prevention of neurotoxic side effects induced by salinomycin.     

The bulge region of HIV-1 TAR RNA binds metal ions in solution

Binding of Mg²⁺, Ca²⁺ and Co(NH₃)₆³⁺ ions to the HIV-1 TAR RNA in solution was analysed by ¹⁹F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for ¹⁹F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg²⁺ and Ca²⁺ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K_d) of 0.9 ± 0.6 and 2.7 ± 1.7 mM, respectively. Argininamide, inducing largest ¹⁹F chemical shifts changes at FU-23, was used as a reference ligand (K_d = 0.3 ± 0.1 mM). In the Pb²⁺-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg²⁺ and Ca²⁺ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb²⁺-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH₃)₆³⁺ > Mg²⁺ > Ca²⁺] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.     

Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration ([Ca²⁺]) gradient and regular oscillations of the cytosolic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region. Whether this [Ca²⁺] gradient and/or [Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca²⁺] oscillation is not. Next, we monitored [Ca²⁺] dynamics in the endoplasmic reticulum ([Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The [Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the [Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.In many flowering plants, a pollen grain that lands on the top surface of a stigma will hydrate and germinate a pollen tube. Following germination, the pollen tube enters the style and grows through the wall of transmitting tract cells on the way to the ovary, where the tube emerges to release the sperm for double fertilization. Therefore, pollen tube growth is essential for reproduction in flowering plants.Since Brewbaker and Kwack (1963) revealed that Ca²⁺ is essential for in vitro pollen tube cultures, the relationship between the Ca²⁺ concentration ([Ca²⁺]) and pollen tube growth has been further examined under in vitro germination culture conditions. Ratiometric ion imaging using fluorescent dye has revealed that the apical domain of a pollen tube grown in vitro contains a tip-focused [Ca²⁺] gradient (Pierson et al., 1994, 1996; Cheung and Wu, 2008) and that the cytoplasmic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region and the growth rate oscillate with the same periodicity (Pierson et al., 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). Therefore, oscillation of the [Ca²⁺]_cyt has been thought to correlate with pollen tube growth. It is not clear, however, whether regular [Ca²⁺]_cyt oscillations in the tip region occur in pollen tubes growing through stigmas and styles.The [Ca²⁺]_cyt is controlled temporally and spatially by transporters in the membranes of intracellular compartments and in the plasma membrane (Sze et al., 2000). Studies using a Ca²⁺-sensitive vibrating electrode revealed Ca²⁺ influx in the tip region of the pollen tube (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Franklin-Tong et al., 2002). Stretch-activated Ca²⁺ channels have been found in the plasma membrane using patch-clamp electrophysiology (Kuhtreiber and Jaffe, 1990; Dutta and Robinson, 2004). Recently, CNGC18 was identified as a Ca²⁺-permeable channel in the plasma membrane that is essential for pollen tube growth (Frietsch et al., 2007). The intracellular compartments that store Ca²⁺ in the pollen tube and the relevant Ca²⁺ transporters, however, have yet to be identified.Yellow cameleons are genetically encoded Ca²⁺ indicators that were developed to monitor the [Ca²⁺] in living cells (Miyawaki et al., 1997). These indicators are chimeric proteins consisting of enhanced cyan fluorescent protein (ECFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and enhanced yellow fluorescent protein (EYFP). When the CaM domain binds Ca²⁺, the domain associates with the M13 peptide and induces fluorescence resonance energy transfer (FRET) between ECFP and EYFP. Several types of cameleons have been developed by tuning the CaM domain binding affinity for Ca²⁺. Yellow cameleon 2.1 (YC2.1) is a high-affinity indicator that has been used to monitor the [Ca²⁺]_cyt in Arabidopsis (Arabidopsis thaliana) guard cells (Allen et al., 1999, 2000, 2001), Lilium longiflorum and Nicotiana tabacum pollen tubes (Watahiki et al., 2004), and the root hair of Medicago truncatula (Miwa et al., 2006). YC3.1 is a low-affinity indicator that has been used to monitor the [Ca²⁺]_cyt during pollen germination and in papilla cells of Arabidopsis (Iwano et al., 2004).Recently, YC3.60 was developed as a new YC variant (Nagai et al., 2004), in which the acceptor fluorophore is a circularly permuted version of Venus rather than EYFP (Nagai et al., 2002). YC3.60 has a monophasic Ca²⁺ dependency with a dissociation constant (K_d) of 0.25 μm. Compared with YC3.1, YC3.60 is equally bright with a 5- to 6-fold larger dynamic range. Thus, YC3.60 results in a markedly enhanced signal-to-noise ratio, thereby enabling Ca²⁺ imaging experiments that were not possible with conventional YCs. On the other hand, YC4.60 was developed by mutating the Ca²⁺-binding loop of CaM in YC3.60. Because YC4.60 has a significantly lower Ca²⁺ affinity with a biphasic Ca²⁺ dependency (K_d: 58 nm and 14.4 μm), it allows changes in [Ca²⁺] dynamics to be detected against a high background [Ca²⁺] (Nagai et al., 2004).To examine whether the [Ca²⁺]_cyt oscillates in pollen tubes growing through a stigma after pollination (in vivo condition), in those placed in germination medium immediately after passing through a style (semi-in vivo condition), or in those grown in germination medium (in vitro condition), we generated transgenic Arabidopsis and N. tabacum lines expressing the YC3.60 gene in their pollen grains and monitored Ca²⁺ dynamics in the pollen tube tip. We also examined how inhibitors of pollen tube growth affect Ca²⁺ dynamics in pollen tubes growing under the semi-in vivo condition. To examine Ca²⁺ dynamics in the endoplasmic reticulum (ER), we generated transgenic Arabidopsis plants expressing YC4.60 in the pollen tube ER. The results are discussed in relation to the physiological relevance of [Ca²⁺] oscillations for pollen tube growth.     

The role of polyamines, Na(+) and K(+) in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine·purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na⁺ and K⁺. In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 10⁹/M range. In contrast to previous reports, K⁺ did not inhibit triplex formation and at 150 mM the binding constants were again in the 10⁹/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K⁺ the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K⁺ was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 µM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K⁺ may not be detrimental. However, it may be difficult to achieve binding constants above ~10⁹/M even in the presence of polycations.     

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.     

Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba

Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca²⁺ channels and cytosolic Ca²⁺ elevation as a link between EPWs and forisome dispersion. Ca²⁺ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca²⁺ of ∼50 nM were independently estimated using Ca²⁺-selective electrodes and a Ca²⁺-sensitive dye. Transient changes in cytosolic Ca²⁺ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca²⁺ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca²⁺ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca²⁺ channel blocker mapped an increased density of Ca²⁺ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca²⁺, release of which depends on both plasma membrane and ER Ca²⁺ channels.     

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.     

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.     

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.     

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4⁺CD25⁺Foxp3⁺ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4⁺ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4⁺CD25⁺Foxp3⁺ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4⁺CD25⁺ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.