Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.     

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.     

Voltage-independent calcium release in heart muscle

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.     

沸石和方解石复合覆盖层控制底泥氮磷释放的效果及机理分析

通过模拟试验，研究了沸石和方解石复合覆盖层控制底泥氮磷释放的效果及机理。结果表明，（1）Ca^2＋会促进方解石对磷酸盐的去除，并且Ca^2＋浓度越高，对磷酸盐去除的促进作用越强，而Na＋则会抑制方解石对磷酸盐的去除。（2）采用CaCl2对天然沸石进行改性，可以提高沸石Ca^2＋的交换量和降低Na^＋的交换量；而采用NaCl改性则可以明显提高沸石Na^＋的交换量，并且大幅度地降低沸石Ca^2＋的交换量。（3）沸石与方解石复合覆盖层不仅可以控制底泥氨氮的释放，而且可以抑制底泥磷的释放，并且复合覆盖层对底泥磷释放的控制效果受沸石改性的影响，控制效果从大到小依次为CaCl：改性沸石〉天然沸石〉NaCl改性沸石。     

Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron

Confocal laser-scanned microscopy and long-wavelength calcium (Ca2+) indicators were combined to monitor both sustained and rapidly dissipating Ca2+ gradients in voltage-clamped sympathetic neurons isolated from the bullfrog. After a brief activation of voltage-dependent Ca2+ channels, Ca2+ spreads inwardly, and reaches the center of these spherical cells in about 300 milliseconds. Although the Ca2+ redistribution in the bulk of the cytosol could be accounted for with a radial diffusion model, local nonlinearities, suggesting either nonuniform Ca2+ entry or spatial buffering, could be seen. After electrical stimulation, Ca2+ signals in the nucleus were consistently larger and decayed more slowly than those in the cytosol. A similar behavior was observed when release of intracellular Ca2+ was induced by caffeine, suggesting that in both cases large responses originate from Ca2+ release sites near or within the nucleus. These results are consistent with an amplification mechanism involving Ca2(+)-induced Ca2+ release, which could be relevant to activity-dependent, Ca2(+)-regulated nuclear events.     

Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. Here, we demonstrate that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner.     

Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells

Inositol 1,4,5-trisphosphate [I(1,4,5)P3] is a second messenger generated along with diacylglycerol upon the binding of various physiological agents with their cell surface receptors. I(1,4,5)P3 mobilizes Ca2+ from intracellular storage sites through a receptor-coupled mechanism, and the subsequent increased intracellular free calcium ion concentration [( Ca2+]i) activates a multitude of cellular responses. Electropermeabilized neoplastic rat liver epithelial (261B) cells were used to study Ca2+ sequestration, a process that reverses the elevated [Ca2+]i to resting levels and replenishes intracellular Ca2+ pools. Although I (1,4,5)P3-mobilized Ca2+ is readily sequestered into storage pools by the action of Ca2+-adenosine triphosphatases, Ca2+ mobilized by addition of the nonmetabolized inositol trisphosphate isomer I(2,4,5)P3 is not sequestered, suggesting that metabolism is necessary to eliminate the stimulus for Ca2+ release. Several inositol phosphate compounds were examined for their ability to lower the buffer [Ca2+] to determine if a specific I(1,4,5)P3 metabolite might be involved in stimulating Ca2+ sequestration; of these, I(1,3,4,5)P4 alone was found to induce Ca2+ sequestration, demonstrating a physiological role for this inositol trisphosphate metabolite.     

Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes

Intracellular calcium (Ca2+) is a ubiquitous second messenger. Information is encoded in the magnitude, frequency, and spatial organization of changes in the concentration of cytosolic free Ca2+. Regenerative spiral waves of release of free Ca2+ were observed by confocal microscopy in Xenopus laevis oocytes expressing muscarinic acetylcholine receptor subtypes. This pattern of Ca2+ activity is characteristic of an intracellular milieu that behaves as a regenerative excitable medium. The minimal critical radius for propagation of focal Ca2+ waves (10.4 micrometers) and the effective diffusion constant for the excitation signal (2.3 x 10(-6) square centimeters per second) were estimated from measurements of velocity and curvature of circular wavefronts expanding from foci. By modeling Ca2+ release with cellular automata, the absolute refractory period for Ca2+ stores (4.7 seconds) was determined. Other phenomena expected of an excitable medium, such as wave propagation of undiminished amplitude and annihilation of colliding wavefronts, were observed.     

The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

The effects of various Ca^2 -modifying drugs on moue egg fertilization were studied.Ca^2 chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca^2 channel bolcker,verspamil,did not have any effect.When intracellular Ca^2 release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca^2 oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca^2 -At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca^2 release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca^2 release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca^2 channel and intracellular release induced by IP3 and not the only pathways for producing Ca^2 transients in moue eggs.     

Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain

Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.     

Ca(2+)-induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose

Calcium-induced calcium release (CICR) may function widely in calcium-mediated cell signaling, but has been most thoroughly characterized in muscle cells. In a homogenate of sea urchin eggs, which display transients in the intracellular free calcium concentration ([Ca2+]i) during fertilization and anaphase, addition of Ca2+ triggered CICR. Ca2+ release was also induced by the CICR modulators ryanodine and caffeine. Responses to both Ca2+ and CICR modulators (but not Ca2+ release mediated by inositol 1,4,5-trisphosphate) were inhibited by procaine and ruthenium red, inhibitors of CICR. Intact eggs also displayed transients of [Ca2+]i when microinjected with ryanodine. Cyclic ADP-ribose, a metabolite with potent Ca(2+)-releasing properties, appears to act by way of the CICR mechanism and may thus be an endogenous modulator of CICR. A CICR mechanism is present in these nonmuscle cells as is assumed in various models of intracellular Ca2+ wave propagation.     

Extracellular and Intracellular Calcium both Involved in the Jasmonic Acid Induced Calcium Mobilization in Arabidopsis thaliana

The objective of this experiment is to evaluate the role of intracellular and extracellular Ca2＋ and calmodulin （CAM） in jasmonic acid （JA） signaling. The laser scanning microscopy was used to detect the changes of [Ca2＋]cyt of Arabidopsis thaliana leaf cells which pretreated with different types of calcium channel blocker. Moreover, the expression of VSP, one of JA response genes, was also investigated after pretreated with the above blocker and antagonist of CaM. The results showed that extracellular and intracellular calcium both involved in the JA-induced Ca2＋ mobilization, and then Ca2＋ exerted its functions through activating the CaM or CaM related proteins. The apoplast calcium influx and the calcium release from the calcium stores are both involved in the JA-induced calcium mobilization, then the JA-induced Ca2＋ transmited the JA signal through CaM or CaM related proteins, and regulated the JA responsive genes.     

Relationship Between Ca2+-CaM and Ethylene-Induced PG Activity in Tomato Fruit

Polygalacturonase (PG) was studied during ripening and senescence of postharvest tomatofruit at pink stage at low and normal temperature. The results showed that the PG activity increased, thendecreased during ripening and senescence of tomato. Low temperature inhibited but ethylene enhanced PGactivity. Ethylene also enhanced caimodulin content, which was dependent on Ca2+ concentration in cell.When EGTA(Ca2+ chelator), verapamil (Vp) and LaCl3 (Ca2+ channel blockers), trifluoperazine and chloro-promaize (two CaM antagonisms) were used to treat tomato fruit at green mature stage with ethylene, theycould reverse ethylene-induced increase in PG activity, but Vp, chloropromaize (CPZ), trifluoperazine(TFP) could not directly influence PG activity, which indirectly indicated that influx of Ca2+ from the ex-tracellular space including the cell wall via the Ca2+ channel localized in plasma membrane and CaM were re-quired for ethylene-induced PG activity increase and that ethylene signal transduction may be related to Ca2+- CaM messenger system.     

Target cell-dependent normalization of transmitter release at neocortical synapses

The efficacy and short-term modification of neocortical synaptic connections vary with the type of target neuron. We investigated presynaptic Ca2+ and release probability at single synaptic contacts between pairs of neurons in layer 2/3 of the rat neocortex. The amplitude of Ca2+ signals in boutons of pyramids contacting bitufted or multipolar interneurons or other pyramids was dependent on the target cell type. Optical quantal analysis at single synaptic contacts suggested that release probabilities are also target cell-specific. Both the Ca2+ signal and the release probability of different boutons of a pyramid contacting the same target cell varied little. We propose that the mechanisms that regulate the functional properties of boutons of a pyramid normalize the presynaptic Ca2+ influx and release probability for all those boutons that innervate the same target cell.     

How synaptotagmin promotes membrane fusion

Synaptic vesicles loaded with neurotransmitters are exocytosed in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent manner after presynaptic depolarization induces calcium ion (Ca2+) influx. The Ca2+ sensor required for fast fusion is synaptotagmin-1. The activation energy of bilayer-bilayer fusion is very high (approximately 40 k(B)T). We found that, in response to Ca2+ binding, synaptotagmin-1 could promote SNARE-mediated fusion by lowering this activation barrier by inducing high positive curvature in target membranes on C2-domain membrane insertion. Thus, synaptotagmin-1 triggers the fusion of docked vesicles by local Ca2+-dependent buckling of the plasma membrane together with the zippering of SNAREs. This mechanism may be widely used in membrane fusion.     

Coupling diurnal cytosolic Ca2+ oscillations to the CAS-IP3 pathway in Arabidopsis

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.     

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.