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1.
We recently reported the first molecular genetic evidence that Dictyostelium Ca2+ responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) – responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca2+-binding proteins in the ER, even though the influx of Ca2+ into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30–70 nM additional Ca2+ to the responses. Here we report that this additional ER contribution to the cytosolic Ca2+ signal depends upon extracellular Ca2+– it does not occur in the absence of extracellular Ca2+, increases to a maximum as the extracellular Ca2+ levels rise to 10 μM and then remains constant at extracellular Ca2+ concentrations up to at least 250 μM. These results suggest that Ca2+ influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca2+-induced Ca2+ release from the ER. By way of contrast, we show that Ca2+ responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca2+. Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca2+.  相似文献   

2.
In contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca2+ the vacuolar ion currents at resting cytosolic Ca2+are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca2+ concentrations. Patch—clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca2+- and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K+ overs C permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca2+, with a Kd≈ 6 µM. At physiological vacuolar Ca2+ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K+ equilibrium potential. It is proposed that transport of K+ through this cation channel controls the electrical potential difference across the tonoplast.  相似文献   

3.
Recent studies have suggested that Ca2+/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca2+ is required for the activation of CaM and CaM-like proteins, the origin of the Ca2+ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba . BL-dependent proton pumping was affected neither by Ca2+ channel blockers nor by changes of Ca2+ concentration in the medium used for the GCPs. Addition of Ca2+ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 m M caffeine, which releases Ca2+ from the intracellular stores, and by 10 μ M 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ) and 10 μ M cyclopiazonic acid (CPA), inhibitors of Ca2+-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μ M thapsigargin, an inhibitor of animal ER-type Ca2+-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca2+ thought to be required for BL-dependent proton pumping may originate from intracellular Ca2+ stores, most likely from ER in guard cells, and that this origin of Ca2+ may generate a stimulus-specific Ca2+ signal for stomatal opening.  相似文献   

4.
Potassium ion channels in the plasmalemma   总被引:2,自引:0,他引:2  
The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K+ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K+ equilibrium potential (EK) – membrane potential (Vm), or secondary active by coupling through a carrier to the inward driving force of H+ or Na+. Known K+ channels are permeable to monovalent cations, a permeability order being K+ > Rb+ > NH4+ > Na+≥ Li+ > Cs+. The macroscopic K+ currents across a cell or protoplast surface commonly show rectification, i.e. a Vm-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca2+, of K+, of H+, or by the K+ driving force. Analysis by the patch clamp technique reveals that plant K+ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K+ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.  相似文献   

5.
Abstract: A charybdotoxin-sensitive, Ca2+-activated K+ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and 86Rb+-influx and efflux experiments. Channel activity was dependent on the presence of Ca2+ on the cytosolic face of the membrane with a threshold concentration of 100 n M . It was inhibited by charybdotoxin (IC50 30 n M ) and quinine (IC50 0.1 m M ) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC50 0.7 n M ) and endothelin-3 (EC50 7–10 n M ). The actions of endothelins were prevented by BQ-123 ( K i = 8 n M ) in a competitive fashion, hence suggesting the involvement of an ETA-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca2+-activated K+ channels for mediating K+ movements across the blood-brain barrier is discussed.  相似文献   

6.
Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K+ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K+ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I A (transient K+ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I K (delayed rectifier K+ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I K current. In contrast, the reversible depolarization and slowly recovering inhibition of I A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K+ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K+ currents partly involved in the control of granule cell death.  相似文献   

7.
The role of a recently identified K+ATP channel in preventing H2O2 formation was examined in isolated pea stem mitochondria. The succinate-dependent H2O2 formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H2O2 formation was not influenced. The succinate-sustained H2O2 generation was also unaffected by nigericin (a H+/K+ exchanger), but completely prevented by valinomycin (a K+ ionophore). In addition, cyclosporin A (a K+ATP channel opener) inhibited this H2O2 formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K+ channel faces the intermembrane space. Finally, the succinate-dependent H2O2 formation was partially prevented by phenylarsine oxide (a thiol oxidant).  相似文献   

8.
Free cytosolic Ca2+ ([Ca2+]cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca2+]cyt elevation is associated with Ca2+-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca2+ channels and their regulation remains limited in planta . A type of voltage-dependent Ca2+-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba2+ and Ca2+, and their activities can be inhibited by micromolar Gd3+. The unitary conductance and the reversal potential of the channels depend on the Ca2+ or Ba2+ gradients across the plasma membrane. The inward whole-cell Ca2+ (Ba2+) current, as well as the unitary current amplitude and NPo of the single Ca2+ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.  相似文献   

9.
Abstract: During K+ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba2+ could substitute for 1 m M Ca2+ in evoking the release of endogenous glutamate. In addition, Ba2+ was found to evoke glutamate release in the absence of K+-induced depolarization. Ba2+ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [3H]-tetraphenylphosphonium cation distribution. Ba2+ partially inhibited the increase in synaptosomal K+ efflux produced by depolarization, as reflected by the redistribution of radiolabeled 86Rb+. The release evoked by Ba2+ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca2+] increased during stimulation by approximately 200 n M , but cytosolic [Ba2+] increased by more than 1 μ M . Taken together, our results indicate that Ba2+ initially depolarizes synaptosomes most likely by blocking a K+ channel, which then activates TTX-sensitive Na+ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca2+channels to evoke neurotransmitter release directly. Though Ba2+-evoked glutamate release was comparable in level to that obtained with K+-induced depolarization in the presence of Ca2+, the apparent intrasynaptosomal level of Ba2+ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca2+.  相似文献   

10.
The SV channel encoded by the TPC1 gene represents a Ca2+- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca2+ signals and cytosolic Ca2+ is required for SV channel function, we here studied the Ca2+-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca2+ sensitivity and Ca2+ impermeability. K+ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca2+ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca2+ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca2+. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca2+ before SV channel activity vanishes and K+ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca2+ signals resulting in overproduction of jasmonate.  相似文献   

11.
The generally rhizotoxic ion Al3+ often enhances root growth at low concentrations. The hypothesis that Al3+ enhances growth by relieving H+ toxicity was tested with wheat seedlings ( Triticum aestivum L.). Growth enhancement by Al3+ only occurred under acidic conditions that reduced root elongation. Al3+ increased cell membrane electrical polarity and stimulated H+ extrusion. Previous investigations have shown that Al3+ decreases solute leakage at low pH and that the alleviation of H+ toxicity by cations appears to be a general phenomenon with effectiveness dependent upon charge (C3+>C2+>Cl+). Alleviation of one cation toxicity by another toxic cation appears to be reciprocal so that Al3+ toxicity is relieved by H+. It has been argued previously that this latter phenomenon accounts for the apparent toxicity of ALOH2+ and Al(OH)+2. Reduction of cell-surface electrical potential by the ameliorative cation may reduce the cell-surface activity of the toxic cation.  相似文献   

12.
The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca2+ signalling by either amplifying Ca2+ entry through voltage-gated Ca2+ channels by Ca2+-induced Ca2+ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca2+ release (IICR). The ER Ca2+ store emerges as a single interconnected pool, thus allowing for a long-range Ca2+ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca2+ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca2+ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca2+ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.  相似文献   

13.
Abstract. For Chlorella emersonii , plausible membrane potentials between –80 and –120 mV were calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and the medium. Furthermore, these calculated membrane potentials were influenced in a way expected from the literature, by different metabolic conditions induced by light or dark, anaerobiosis, glucose, and by inhibition or uncoupling of electron transport.
Nevertheless, the experiments presented here indicate that TPP+ is unsuitable as a probe for electrical potentials, at least in Chlorella emersonii. The reasons for this conclusion are as follows:
  • 1. 

    Much of the incorporated TPP+-14C could not be exchanged against unlabelled TPP+.

  • 2. 

    The uptake of TPP+-14C was very slow and exhibited complex rather than simple saturation kinetics.

  • 3. 

    A large adsorption of TPP+-14C took place even after the cells were killed; the adsorption by living cells was only 20–60% higher than with killed cells. Furthermore, the adsorption by killed cells showed kinetics similar to living cells.

  相似文献   

14.
Ecosystem flux measurements using the eddy covariance (EC) technique were undertaken in 4 subsequent years during summer for a total of 562 days in an arctic wet tundra ecosystem, located near Cherskii, Far-Eastern Federal District, Russia. Methane (CH4) emissions were measured using permanent chambers. The experimental field is characterized by late thawing of permafrost soils in June and periodic spring floods. A stagnant water table below the grass canopy is fed by melting of the active layer of permafrost and by flood water. Following 3 years of EC measurements, the site was drained by building a 3 m wide drainage channel surrounding the EC tower to examine possible future effects of global change on the tundra tussock ecosystem. Cumulative summertime net carbon fluxes before experimental alteration were estimated to be about +15 g C m−2 (i.e. an ecosystem C loss) and +8 g C m−2 after draining the study site. When taking CH4 as another important greenhouse gas into account and considering the global warming potential (GWP) of CH4 vs. CO2, the ecosystem had a positive GWP during all summers. However CH4 emissions after drainage decreased significantly and therefore the carbon related greenhouse gas flux was much smaller than beforehand (475 ± 253 g C-CO2-e m−2 before drainage in 2003 vs. 23 ± 26 g C-CO2-e m−2 after drainage in 2005).  相似文献   

15.
In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5 mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 μM Zn2+. A znuA–lacZ operon fusion was repressed by 5 μM Zn2+ and showed a more than 20-fold increase in β-galactosidase activity when Zn2+ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .  相似文献   

16.
Abstract. Rates of proton extrusion and potassium (86Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K+ concentrations (0.01 to 1 mol m−3), H+ efflux and K+ influx were strongly correlated in both low- and high-K+ roots, although K+: H+ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m−3), H+ efflux was either reduced or remained unchanged while K+ influxes increased. In the presence of Na2SO4, rates of H+ extrusion demonstrated similar cation dependence, although below 10 mol m−3 Na2SO4, H+ fluxes were generally 50% lower than in equivalent concentrations of K2SO4. These observations are considered in the context of current hypotheses regarding the mechanisms of k+/H+ exchange.  相似文献   

17.
Abstract: The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura-2 imaging. Following KCI depolarization, addition of the protonophore carbonylcyanide m -chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP-releasable pool was found in nondepolarized cells. Although the KCI-evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCI depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine-123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP-releasable pool was found subsequent to KCI depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCI-evoked Ca2+ elevation is decreased. It is concluded that the CCCP-induced increase in cytoplasmic Ca2+ response to KCI is due to inhibition of nonmitochondrial ATP-dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage-activated Ca2+ channel activity.  相似文献   

18.
Irradiation of cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells with ultraviolet light caused of loss of K+, which occurred with sigmoid kinetics. The kinetics of loss of K+ were not changed when the extracellular concentration of K+ was held constant during the period of efflux. Furthermore, the rate of loss of K+ was approximately the same even though the K+ concentration in the medium was increased from 0.1 to 10 m M . The kinetics of uptake of the lipophilic methyltriphenylphosphonium cation, an indicator of the plasma membrane potential, were linear throughout the period of K+ efflux, suggesting that the starting and stopping of K+ efflux do not reflect a passive response to changes in the membrane potential of the cells. The results are interpreted in terms of activation and inactivation of an efflux channel or pump for K+.  相似文献   

19.
Characterization of the adsorption process by the phages hv and ATCC 15807-B1 to Lactobacillus helveticus ATCC 15807 was carried out. For this purpose, the influence of Ca2+ ions, temperature and physiological cell state were studied. The ability of several saccharides and related compounds to inactivate the phages hv and ATCC 15807-B1 was determined to investigate their potential role as phage receptors. Furthermore, several chemical treatments on the sensitive strain cells were carried out to study their influence on phage adsorption. Cell lysis and plaque formation were independent of Ca2+ ions for phage hv, but the cation was indispensable for completion of the lytic cycle of phage ATCC 15807-B1. However, for this phage, Ca2+ was not necessary for the adsorption process. The adsorption rates were almost normal for both phages within the temperature range examined (0 – 50 °C) and the adsorption kinetics were practically identical on viable and non-viable cells. The saccharides and related compounds used did not produce inactivation of the phages, suggesting that they were not essential components of phage receptor structures. Lactobacillus helveticus ATCC 15807 cells treated with SDS 1%, SDS 0·5% -EDTA 50 mmol l−1 or NaOH 50 mmol l−1 exhibited reduced adsorption of the phages, indicating possible damage or extraction of receptors from the cell wall. Phage adsorption presents an extremely attractive target for interfering in the lytic cycle of phages.  相似文献   

20.
Outer membrane porin protein of Campylobacter jejuni   总被引:1,自引:0,他引:1  
Abstract Protein e, a 43-kDa protein from the outer membrane of Campylobacter jejuni UA580, was purified and reconstituted into lipid bilayer membranes. It was shown to form small channels with a single channel conductance of 8.82 nS in 1M KCl. Zero current potential measurements demonstrated that the channel was approx. 10-fold selective for K+ over Cl ions. A porin with a similar single channel conductance was observed in fractions from the outer membrane of Campylobacter fetus UA60.  相似文献   

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