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Radiation therapy efficiently eliminates cancer cells and reduces tumor growth. To understand collateral agonistic and antagonistic effects of this treatment on the immune system, we examined the impact of x-ray irradiation on human T cells. We find that, in a major population of leukemic Jurkat T cells and peripheral blood mononuclear cells, clinically relevant radiation doses trigger delayed oscillations of the cytosolic Ca2+ concentration. They are generated by store-operated Ca2+ entry (SOCE) following x-ray-induced clustering of Orai1 and STIM1 and formation of a Ca2+ release-activated Ca2+ (CRAC) channel. A consequence of the x-ray-triggered Ca2+ signaling cascade is translocation of the transcription factor nuclear factor of activated T cells (NFAT) from the cytosol into the nucleus, where it elicits the expression of genes required for immune activation. The data imply activation of blood immune cells by ionizing irradiation, with consequences for toxicity and therapeutic effects of radiation therapy., (© 2022 Tandl et al.)

Ca 2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca 2+ concentration upon stimulation. The elevated Ca 2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca 2+ at different locations in the cell. Ca 2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca 2+ ions. A major function of the rise in intracellular Ca 2+ is the change of the genetic expression pattern of the cell via the activation of Ca 2+ -responsive transcription factors. It has been proposed that Ca 2+ -responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca 2+ . Moreover, it has been suggested that the mode of entry determines whether an influx of Ca 2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca 2+ induces an intracellular signaling cascade, involving the activation of the Ca 2+ /calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca 2+ /calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca 2+ concentration in the cytoplasm and the nucleus, the role of Ca 2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca 2+ signaling and gene expression.

The TRPM8 cation channel can be activated by the cooling compound icilin. Recently, we showed that stimulation of TRPM8 channels induces a signaling cascade leading to the activation of the transcription factor AP-1. Additionally, expression of the AP-1 constituent c-Fos has been shown to be induced following TRPM8 stimulation. c-Fos is frequently used as a marker for neuronal activity. Here, we have analyzed the mechanism connecting TRPM8 stimulation and c-Fos expression. Furthermore, we analyzed the expression of the neuronal activity-responsive transcription factor Egr-1 following TRPM8 activation. The results show that icilin-induced stimulation of TRPM8 channels increased c-Fos promoter activity and induced c-Fos expression. Moreover, icilin stimulation increased Egr-1 promoter activity and induced the expression of Egr-1. Pharmacological inhibition of TRPM8 blocked the icilin-induced expression of Egr-1 and c-Fos. An influx of Ca 2+ ions into the cells via TRPM8 was necessary to stimulate Egr-1 and c-Fos expression following icilin treatment. Genetic experiments revealed that serum response elements within the Egr-1 and c-Fos promoters are crucial to couple TRPM8 stimulation with enhanced transcription of both the Egr-1 and c-Fos genes. These data were corroborated by experiments showing that TRPM8 stimulation increased the transcriptional activation potential of Elk-1, a SRE binding protein. c-Fos is important for neuronal excitability and survival. Egr-1 plays an important role in synaptic plasticity, consolidation and reconsolidation of long-term memory. Elk-1 may preserve neurons against toxic insults but may also induce depressive behaviour. The fact that TRPM8 stimulation activates the transcription factors c-Fos, Egr-1, and Elk-1 connects TRPM8 signaling with maintaining important brain functions., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Measurements of the membrane capacitance on animal cells has provided an excellent technique for monitoring of exo- and endocytotic activity in intact living cells. Here we review recent data in which the same technique was applied to plant cells and cells of the budding yeast Saccharomyces cerevisiae. The data show that unitary exo- and endocytotic events can also be measured with the same technique after removing the cell wall from these cells. The resulting protoplasts execute the same type of transient and permanent fusion/fission that is known from animal cells. Also the size of the vesicles, which are fusing or budding, are of the same order of magnitude as those recorded in animal cells. Together these data support the view of an evolutionary conserved mechanism for unitary exo- and endocytosis events in eukaryotes. The successful recordings of exo- and endocytotic activity in Saccharomyces cerevisiae by capacitance measurements now pave the way for correlating the abundant information on the molecular machinery of exo- and endocytosis in this model organism with distinct functional properties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The steroid pregnenolone sulfate activates the transcription factor activator protein-1 (AP-1) via stimulation of transient receptor potential melastatin-3 (TRPM3) channels. Here, we show that the signaling pathway requires an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) levels. The upregulation of AP-1 was attenuated in cells that overexpressed mitogen activated protein kinase phosphatase-1, indicating that Ca(2+) ions prolong the signaling cascade via activation of mitogen activated protein kinases. On the transcriptional level, expression of a dominant-negative mutant of the basic region leucine zipper protein c-Jun, a major constituent of the AP-1 transcription factor complex, or expression of a c-Jun-specific short hairpin RNA attenuated pregnenolone sulfate-induced AP-1 activation. In addition, stimulation of TRPM3 channels increased the transcriptional activation potential of the basic region leucine zipper protein ATF2. Inhibition of ATF2 target gene expression via expression of a dominant-negative mutant of ATF2 or expression of an ATF2-specific short hairpin RNA interfered with TRPM3-mediated stimulation of AP-1. Moreover, we show that a dominant-negative mutant of the ternary complex factor (TCF) Elk-1 attenuated the upregulation of AP-1 following stimulation of TRPM3 channels. Thus, c-Jun, ATF2, and TCFs are required to connect the intracellular signaling cascade elicited by activation of TRPM3 channels with enhanced transcription of AP-1-regulated genes. We conclude that pregnenolone sulfate-induced TRPM3 channel activation changes the gene expression pattern of the cells by activating transcription of c-Jun-, ATF2-, and TCF-controlled genes., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Signals recorded at the cell membrane are meaningful indicators of the physiological vs. pathological state of a cell and will become useful diagnostic elements in nanomedicine. In this project we present a coherent strategy for the design and fabrication of a bio-nano-sensor that monitors changes in intracellular cell calcium concentration and allows an easy read out by converting the calcium signal into an electrical current in the range of microampere that can be easily measured by conventional cell electrophysiology apparatus.

Single-channel current-voltage (IV) curves of human large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels are quite linear in 150 mM KCl. In the presence of Ca(2+) and/or Mg(2+), they show a negative slope conductance at high positive potentials. This is generally explained by a Ca(2+)/Mg(2+) block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca(2+) and 2.5 mM Mg(2+) per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca(2+)/Mg(2+). In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca(2+)/Mg(2+). The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.

Elevated extracellular Ca(2+) concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.

Here we examine the potential coupling between the synthesis of secretory proteins and the sensitivity of exocytosis to the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)) in plant cell. We therefore monitor in tobacco protoplasts the excursion of the membrane capacitance in response to an elevation of [Ca(2+)](i) as a measure for exocytotic activity. The data show that a ramp like elevation of [Ca(2+)](i) generates in protoplasts from wild type plants and from transgenic plants, which overexpress the secreted α-amylase, an exocytotic burst with an initial steep and a subsequent slow phase. The largest capacitive burst is obtained in α-amylase producing plants and the amplitude of the [Ca(2+)](i) evoked C(m) excursion is a function of the amylase synthesis of the plants. The data support a model according to which plant cells have at least two serial [Ca(2+)](i) sensitive processes in the final steps of their exocytotic pathway. The overproduction of a secreted cargo does not affect the kinetics of this process but the number of vesicles in pools upstream of the [Ca(2+)](i) sensitive steps., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glucose stimulation induces expression of the transcription factors Egr-1 and c-Jun as well as phosphorylation of CREB and c-Jun in insulinoma cells. The activator protein-1 (AP-1) binding sites within the c-Jun promoter, the serum response elements (SREs) within the Egr-1 promoter, and cyclic AMP response elements (CREs) function as glucose responsive element. Glucose-induced transcriptional regulation was attenuated by inhibition of L-type Ca(2+) channels, or chelating cytoplasmic Ca(2+). It has been proposed that a rise in nuclear Ca(2+) is required for CREB-mediated transcription of CRE-regulated genes, while elevated cytoplasmic Ca(2+) levels trigger an upregulation of SRE-containing genes. Here, we show that a rise in cytoplasmic Ca(2+) is required for AP-1, CRE, and SRE mediated transcription in glucose-stimulated insulinoma cells. Buffering Ca(2+) in the nucleus or the endoplasmic reticulum had no inhibitory effect upon transcription. However, overexpression of the mitochondrial protein Fis-1 or inhibition of the GTPase Drp-1 impaired glucose-stimulated gene transcription in insulinoma cells, suggesting that the mitochondria play an important role in regulating Ca(2+) mediated gene transcription. The extracellular signal-regulated protein kinase functions as an essential link connecting glucose stimulation, the rise in cytoplasmic Ca(2+), and enhanced transcription in insulinoma cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Our aim was to get new information about the Peroxisome Proliferator Activated Receptor gamma (PPARγ)-independent pathway involved in the antiproliferative action of PPARγ ligands in breast cancer cells. We investigated the effects of Troglitazone (TGZ), Ciglitazone (CGZ), Rosiglitazone (RGZ) and, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ(2)) on the hormone-dependent breast cancer cell line MCF7. The early transcription factor EGR1 (Early Growth Response gene 1) mRNA and protein levels peaked after 3h of incubation with 25μM TGZ, CGZ or 15d-PGJ(2) and then gradually decreased. RGZ, the most potent activator of PPARγ, did not show this effect. The PPARγ antagonist GW 9662 did not block EGR1 mRNA induction which also still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ. EGR1 mRNA induction required ERK1/2 phosphorylation which was not blocked by EGF Receptor (EGFR) inhibition. The ERK1/2 pathway was also involved in Δ2-TGZ-induced EGR1 mRNA expression in the hormone-independent breast cancer cell line MDA-MB-231. Using the fluorescent dye Fura2, we showed in MCF7 that TGZ or Δ2-TGZ induced an immediate increase in cytosolic calcium which was required for ERK1/2 phosphorylation and EGR1 mRNA induction as demonstrated by calcium chelation experiments. Furthermore, in MCF7 transfected with siRNA targeting EGR1, Δ2-TGZ inhibited less efficiently cell proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca(2+) ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca(2+)-binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca(2+) (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca(2+) channels (nifedipine). Hence, elevated levels of intracellular Ca(2+), released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargin-induced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.

Calcium-transporting ATPases (Ca(2+) pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca(2+) pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca(2+) pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca(2+) pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a (32)P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca(2+), thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca(2+)-transporting ATPase encoded by a virus.

The transcriptional activity of AP-1 has been analyzed in glucose-stimulated INS-1 insulinoma cells using a chromosomally embedded AP-1-responsive reporter gene. We show that AP-1 activity was significantly elevated in glucose-treated INS-1 cells. Preincubation of the cells with nifedipine or expression of the Ca(2+) binding protein parvalbumin in the cytoplasm of INS-1 cells reduced AP-1 activity. Thus, activation of L-type Ca(2+) channels and an elevated cytoplasmic Ca(2+) concentration are crucial to connecting glucose stimulation with enhanced AP-1 activity. Expression of dominant negative forms of A-Raf, MKK4 or MKK6 and pharmacological inhibition of MEK and p38 revealed that extracellular signal-regulated protein kinase, p38 and c-Jun NH(2)-terminal protein kinase participate in the upregulation of AP-1 activity. Expression of dominant-negative mutants of c-Jun and Elk-1 reduced AP-1 transcriptional activity in INS-1 cells indicating that c-Jun and ternary complex factors are involved in the regulation of AP-1 activity in glucose-stimulated insulinoma cells., ((c) 2010 Wiley-Liss, Inc.)

The biosynthesis of the zinc finger transcription factor Egr-1 is stimulated by many extracellular signaling molecules including hormones, neurotransmitters, growth and differentiation factors. The Egr-1 gene represents a convergence point for many intracellular signaling cascades. An increase of the intracellular Ca(2+) concentration, by activating ionotropic or Galpha(q/11)-coupled receptors or voltage-gated L-type Ca(2+) channels, is often the prerequisite for enhanced Egr-1 gene transcription. This increase has been observed following stimulation with extracellular signaling molecules including ATP, glutamate, thrombin, carbachol, gonadotropin-releasing hormone, or glucose. Egr-1 is thus a Ca(2+) regulated transcription factor - similar to CREB, NFAT, NF-kappaB and others. This review also discusses the importance of the cytoplasmic and nuclear Ca(2+) concentration in transcriptional regulation of the Egr-1 gene., (2010 Elsevier Ltd. All rights reserved.)

Background: The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts., Results: Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK). Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not., Conclusion: This study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

Glucose induces many changes in the transcriptional pattern of beta-cells derived from the endocrine pancreas. The zinc finger protein Egr-1 belongs to the transcription factors that are activated in glucose-treated beta-cells. Egr-1 expression is additionally induced by treatment of MIN6 pancreatic beta-cells with tolbutamide, a compound that triggers a closure of ATP-dependent potassium channels, K(ATP), in the plasma membrane or by KCl that depolarizes the cell membrane. Stimulation with glucose, tolbutamide or KCl induces a Ca2+ influx into the beta-cells via L-type Ca2+ channels. Accordingly, incubation of the cells with the L-type Ca2+ channel blocker nifedipine or the acetoxymethylester of the cytosolic Ca2+ chelator BAPTA prevented Egr-1 expression. Moreover, diacylgycerol-dependent protein kinase C isoenzymes and activation of extracellular signal-regulated protein kinase (ERK) are required for glucose-, tolbutamide- and KCl-induced Egr-1 expression. The signaling cascade was blocked by MAP kinase phosphatase-1 (MKP-1) overexpression that dephosphorylated ERK in the nucleus. Stimulation of beta-cells by glucose, tolbutamide and KCl induced the phosphorylation of the transcription factors Elk-1 and CREB. ChIP experiments revealed that phosphorylated Elk-1 and CREB bound under physiological conditions to the Egr-1 gene. Lentiviral-mediated expression of dominant-negative mutants of Elk-1 or CREB interfered with glucose-, tolbutamide- and KCl-induced upregulation of Egr-1 biosynthesis. Together, these data indicate that stimulus-induced transcription of the Egr-1 gene in beta-cells requires combinatorial regulation by Elk-1 and CREB following activation of ERK. The newly synthesized Egr-1 is biologically active and binds under physiological conditions to the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, transforming growth factor beta and PTEN.

Intracellular calcium is an important second messenger that regulates many cell functions. Recent studies have shown that calcium ions can also regulate the cellular responses to ionizing radiation. However, previous data are restricted to cells treated with low-LET radiations (X rays, gamma rays and beta particles). In this work, we investigated the calcium levels in cells exposed to heavy-ion radiation of high LET. The experiments were performed at the single ion hit facility of the GSI heavy-ion microprobe. Using a built-in online calcium imaging system, the intracellular calcium concentrations were examined in HeLa cells and human foreskin fibroblast AG1522-D cells before and after irradiation with 4.8 MeV/nucleon carbon or argon ions. Although the experiment was sensitive enough to detect the calcium response to other known stimuli, no response to heavy-ion radiation was found in these two cell types. We also found that heavy-ion radiation has no impact on calcium oscillation induced by hypoxia stress in fibroblast cells.

Kinetic investigations of stimulus response coupling in the green alga Chara have revealed that an intermediate second messenger is formed in the process of membrane excitation. This second messenger links electrical stimulation to the mobilization of Ca2+ from internal stores. In the present work, the experimentally based kinetic model, which describes the stimulus-dependent production of the second messenger and Ca2+ mobilization, is combined with a model for inositol 1,4,5-trisphosphate (IP3)-and Ca2+-sensitive gating of a Ca2+-release channel in endomembranes of animal cells. The combination of models allows a good simulation of experimental data, including the all-or-none-type dependence of the Ca2+ response on stimulus duration and complex phase locking phenomena for the dependence of the Ca2+ response on stimulation frequency. The model offers a molecular explanation for the refractory phenomenon in Chara, assigning it to the life time of an inactive state of the Ca2+-release channel. The model furthermore explains the steep dependence of excitation on strength/duration of electrical stimulation as a consequence of an interplay of the dynamical variables in the model.

Electrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)). The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt). When the strength of a pulse exceeded a narrow threshold (suprathreshold pulse) it evoked the full extent of the Ca(2)+(cyt) elevation. This suggests an all-or-none mechanism for Ca(2)+ mobilization. A transient calcium rise could also be induced by one subthreshold pulse if it was after another subthreshold pulse of the same kind after a suitable interval, i.e., not closer than a few 100 ms and not longer than a few seconds. This dependency of Ca(2)+ mobilization on single and double pulses can be simulated by a model in which a second messenger is produced in a voltage-dependent manner. This second messenger liberates Ca(2)+ from internal stores in an all-or-none manner once a critical concentration (threshold) of the second messenger is exceeded in the cytoplasm. The positive effect of a single suprathreshold pulse and two optimally spaced subthreshold pulses on Ca(2)+ mobilization can be explained on the basis of relative velocity for second messenger production and decomposition as well as the availability of the precursor for the second messenger production. Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.

Ca2+ and osmotic driven extension of the surface area of maize coleoptile protoplasts was investigated using capacitance measurements and photolysis of the caged compound DM-nitrophen. Protoplasts responded to an elevation of cytoplasmic Ca2+ (Ca(i)) with a rapid burst in capacitance reaching a maximal increase of 1.3+/-1.1% over the resting cell capacitance. Subsequent lowering of the osmotic potential in the external medium by 210 mosmol caused a further increase in Cm by 26+/-6%. These data indicate two independent pathways for insertion of membrane into the plasma membrane. One is driven by Ca(i) and recruits membrane from a small pool. The osmotic evoked rise in surface area draws membrane from a much larger reservoir and may be driven by membrane tension.

Neomycin and U73122 are known to suppress inositol 1,4,5-trisphosphate (InsP3) production by inhibition of phospholipase C. We studied the effects of these inhibitors on the excitatory currents, Iex, in Chara corallina under voltage-clamp conditions. Computer simulations of the experimental effects by a minimum model for the excitatory reaction pathway allow the assignment of the inhibitory effects to one specific reaction step, i.e. the release of Ca2+ from InsP3-sensitive internal stores. In contrast, the inhibitory effect of La3+ on Iex suggests inactivation of Cl- channels. Furthermore, ryanodine-sensitive Ca2+ stores seem to be irrelevant for electrical excitation in Chara.

Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca(2+)-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca(2+)-binding and -release, respectively (rate constants >> 10(3) sec-1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10(3) sec-1). With 10 mM K+ and 1 mM Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of -105 +/- 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mM Ca2+) and for conductance (1.2 mM Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.

Dual-excitation confocal laser scanning microscopy (CLSM) was used to image the pH-indicator, BCECF, iontophoretically microinjected into stomatal guard cells of Vicia faba during challenge with peptides derived from hydrophilic domains of the maize auxin-binding protein. Only the peptide corresponding to the C-terminal end (Pz151-163) caused significant changes in cytosolic pH, stimulating rapid alkalinisation of 0.4 +/- 0.1 pH units. Cytosolic pH was clamped using the permeant weak acid, butyrate, and this treatment buffered the peptide evoked alkalinisation. In concert with the electrical events monitored at the plasma membrane using whole-cell voltage clamp, this provides strong evidence for a role of [H+] as a signal intermediate in the guard cell transduction network. In preliminary experiments using single-wavelength imaging of the calcium-indicator, Fluo-3, Pz151-163 also stimulated rapid, reversible increases in cytosolic calcium, whilst two other peptides tested had no effect.

RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli--including light, osmotic stress and the phytohormone indoleacetic acid--and InsP3 activates Ca2+ channels and Ca2+ flux across plant vacuolar and microsomal membranes. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements may be controlled by cytoplasmic Ca2+. So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-1-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3) reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels. The data are consistent with a transient rise in cytoplasmic free Ca2+ and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+.

Calmodulin (CaM), a small acidic protein, is one of the best characterised Ca2+ sensors in eukaryotes. This Ca2+-regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellular Ca2+ activity that could initiate adaptive responses under adverse conditions. We report the first molecular cloning and characterisation of a calmodulin gene, VcCaM1 ( Vaccinium corymbosum Calmodulin 1), in the woody shrub, highbush blueberry. VcCaM1 was first identified as VCAL19, a gene induced by aluminium stress in V. corymbosum L. A full-length cDNA of VcCaM1 containing a 766-bp open reading frame (ORF) encoding 149 amino acids was cloned from root RNA. The sequence encodes four Ca2+-binding motifs (EF-hands) and shows high similarity (99%) with the isoform CaM 201 of Daucus carota. Expression analyses showed that following Al treatment, VcCaM1 message level decreased in roots of Brigitta, an Al-resistant cultivar, and after 48 h, was lower than in Bluegold, an Al-sensitive cultivar. VcCAM1 message also decreased in leaves of both cultivars within 2 h of treatment. Message levels in leaves then increased by 24 h to control levels in Brigitta, but not in Bluegold, but then decreased again by 48 h. In conclusion, VcCaM1 does not appear to be directly involved in Al resistance, but may be involved in improved plant performance under Al toxicity conditions through regulation of Ca2+ homeostasis and antioxidant systems in leaves. [ABSTRACT FROM AUTHOR]

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]