Persistent Calcium Inward Current in Internally Perfused Snail Neuron

The inactivation process of the calcium current (ICa) was investigated in a molluscan neuron which was perfused intracellularly and voltage-clamped using a suction pipette technique. The decay phase of the ICa contained a very slowly inactivated component (persistent inward current; PIC). The decay time constant of this component was over 10 sec. An increase in the amplitude of the ICa or the intracellular Ca2+ concentration caused a decrease in the decay time constant of the PIC. Replacing Ba2+ with extracellular Ca2+ increased the decay time constant of the PIC. The differences in the amplitude and the decay kinetics between the ICa and the IBa resulted from changes in the amplitude and the decay time constant of the PIC. These observations support the conclusion that the inactivation of the PIC is calcium dependent [Chad, J., Eckert, R., and Ewald, D. (1984). J. Physiol. (Lond.) 347:279-300].     

Maintenance of Acidic Lateral Intercellular Spaces by Endogenous Fixed Buffers in MDCK Cell Epithelium

The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing solutions was altered. In addition, the divalent cation Sr²⁺ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS. Received: 9 April 1998/Revised: 28 July 1998     

Structure Dependence of the Calcium Dynamics in Purkinje Neuron Dendrites during Generation of Bursting Discharges: a Simulation Study

In the model of a cerebellar Purkinje neuron with reconstructed active dendrites, we investigated the impact of the ratio between volumes of the endoplasmic reticulum (organellar calcium store) and cytosol on the Ca²⁺ dynamics in asymmetrical parts of the dendritic arborization during generation of different structure-dependent patterns of bursting activity. Tonic synaptic excitation homogeneously distributed over the dendrites (a spatially homogeneous stationary input signal) caused spatially heterogeneous variations of the dendritic membrane potential (MP) accompanied by periodical or nonperiodical bursts of action potentials at the cell output. The MP waveforms recorded from the segments of asymmetrical dendrites were then applied to the membrane of selected dendrite segments as command voltages in a dynamic clamp mode. In these segments, the relative size of the stores was varied. This provided equal to each other local calcium currents and influxes into the cytosol of the segment differently filled with the organellar store. Regardless of the impulse pattern, microgeometry of the segment and the store modulated calcium transients exactly in the same way as in previous studies of electrical and concentration responses to local phasic synaptic excitation of the modeled neuron. Peak values of depolarization-induced elevations of the cytosolic Ca²⁺ concentration increased with the portion of the intracellular volume occupied by the store. The most important factor defining this dependence was the ratio of the membrane area vs the organelle-free cytosol volume of the dendritic segment. Concentrations of Са²⁺ deposited in equal-sized segments of asymmetrical parts of the dendritic arborization where asynchronous unequal variations of the MP were observed during generation of nonperiodical bursting at the output demonstrated considerable specificity. A greater amount of calcium was deposited in the segments staying, on average, in a high-depolarization state for a longer time (this intensified activation of calcium channels and amplified the corresponding Ca²⁺ influx into the cytosol). Hence, local dynamics of the Ca²⁺ concentration depend directly on local microgeometry and indirectly on global macrogeometry of the dendrite arborization, as the latter determines spatial asymmetry-related unequal transients in different parts of the dendritic arborization having active membrane properties.     

Movement of Endogenous Calcium in the Elongating Zone of Graviresponding Roots of Zea mays

Endogenous calcium (Ca) accumulates along the lower side ofthe elongating zone of horizontally oriented roots of Zea mayscv. Yellow Dent. This accumulation of Ca correlates positivelywith the onset of gravicurvature, and occurs in the cytoplasm,cell walls and mucilage of epidermal cells. Corresponding changesin endogenous Ca do not occur in cortical cells of the elongatingzone of intact roots. These results indicate that the calciumasymmetries associated with root gravicurvature occur in theoutermost layers of the root Calcium, corn, gravitropism (root), Zea mays     

盐胁迫下外源钙对草莓内源激素含量的影响

以‘达赛莱克特’草莓为试验材料,研究了盐胁迫条件下外源硝酸钙对草莓生理特性的影响。结果表明:Ca(NO3)2处理可抑制NaCl胁迫下草莓叶片细胞膜透性的增加,增加叶片脯氨酸含量,降低根系和叶片中的脱落酸(ABA)含量,增加赤霉素(GA)、生长素(IAA)、玉米素(ZT)的含量和ZT/ABA比值,草莓的盐害指数也有所降低。钙调素拮抗剂W7和三氟拉嗪(TFP)处理能够抑制Ca(NO3)2的作用,且W7的抑制作用比TFP更强。     

Changes in the Spontaneous Calcium Oscillations for the Development of the Preconditioning-Induced Ischemic Tolerance in Neuron/Astrocyte Co-culture

Spontaneous Ca²⁺ oscillations are believed to contribute to the regulation of gene expression. Here we investigated whether and how the dynamics of Ca²⁺ oscillations changed after sublethal preconditioning (PC) for PC-induced ischemic tolerance in neuron/astrocyte co-cultures. The frequency of spontaneous Ca²⁺ oscillations significantly decreased between 4 and 8 h after the end of PC in both neurons and astrocytes. Treatment with 2-APB, an inhibitor of IP3 receptors, decreased the oscillatory frequency, induced ischemic tolerance and a down-regulation of glutamate transporter GLT-1 contributing to the increase in the extracellular glutamate during ischemia. The expression of GLT-1 is known to be up-regulated by PACAP. Treatment with PACAP38 increased the oscillatory frequency, and antagonized both the PC-induced down-regulation of GLT-1 and ischemic tolerance. These results suggested that the PC suppressed the spontaneous Ca²⁺ oscillations regulating the gene expressions of various proteins, especially of astrocytic GLT-1, for the development of the PC-induced ischemic tolerance.     

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25^+/− mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.     

Simulation Study of Non-Organellar Binding of Calcium by Fast and Slow Buffers in Dendrites Containing an Organellar Store

Dependences of intracellular calcium signals on the concentrations of endogenous buffers (slow, parvalbumin, and fast, calmodulin) and a calcium-sensitive fluorophore (Fura-4F) were investigated on mathematical models of compartments of the reconstructed dendrite of a cerebellum Purkinje neuron. A Ca²⁺-storing cistern of the endoplasmic reticulum (ER) was present in the dendrite. Calcium signals developed when the neuron generated responses to single synaptic excitation or intrinsic non-periodical impulse activity. The dynamics of the buffer binding capacity were also studied; this capacity was characterized by the ratio of concentrations of bound and free calcium or concentration increments of the latter. The plasma membrane of the dendrite possessed ion channels (including those of synaptic currents) and the calcium pump characteristic of the mentioned neuron. Model equations took into account Ca²⁺ exchange between the cytosol, buffers, ER, and extracellular medium, as well as diffusion processes. The ER membrane contained the calcium pump, leakage channels, and channels of calcium-induced release and inositol-3-phosphate-dependent releases of Ca²⁺. The ER cistern occupied 1 to 36% of the intracellular volume. Upon different occupancies of the dendrite by the organelle store, an increase in the concentration of the slow buffer insignificantly decreased the cytosolic Ca²⁺ transients with no effect on their shape. The fast buffer and the dye with similar kinetic properties caused slowing down of the rising phase of Ca²⁺ transients, decrease in the early component, and increase in the late component of the latter. In the case of nonperiodical and asynchronous intrinsic oscillations of the membrane potential typical of asymmetrical active dendrites, the slow buffer, like the ER store, bound more Ca²⁺ in compartments of compatible sizes and fillings by the organelles belonging to those metrically asymmetrical branches, which, on average, stayed longer in the state of high depolarization; this provided a greater Ca²⁺ entry from outside. Hence, the pattern of structural/functional organization of calcium signalization in the dendrites can be complemented in the part of both the direct influences of local microgeometry of the dendrite and the indirect ones related to global macrogeometry of the dendritic arborization.     

Buffering Capacity Explains Signal Variation in Symbiotic Calcium Oscillations

Legumes form symbioses with rhizobial bacteria and arbuscular mycorrhizal fungi that aid plant nutrition. A critical component in the establishment of these symbioses is nuclear-localized calcium (Ca2+) oscillations. Different components on the nuclear envelope have been identified as being required for the generation of the Ca2+ oscillations. Among these an ion channel, Doesn''t Make Infections1, is preferentially localized on the inner nuclear envelope and a Ca2+ ATPase is localized on both the inner and outer nuclear envelopes. Doesn''t Make Infections1 is conserved across plants and has a weak but broad similarity to bacterial potassium channels. A possible role for this cation channel could be hyperpolarization of the nuclear envelope to counterbalance the charge caused by the influx of Ca2+ into the nucleus. Ca2+ channels and Ca2+ pumps are needed for the release and reuptake of Ca2+ from the internal store, which is hypothesized to be the nuclear envelope lumen and endoplasmic reticulum, but the release mechanism of Ca2+ remains to be identified and characterized. Here, we develop a mathematical model based on these components to describe the observed symbiotic Ca2+ oscillations. This model can recapitulate Ca2+ oscillations, and with the inclusion of Ca2+-binding proteins it offers a simple explanation for several previously unexplained phenomena. These include long periods of frequency variation, changes in spike shape, and the initiation and termination of oscillations. The model also predicts that an increase in buffering capacity in the nucleoplasm would cause a period of rapid oscillations. This phenomenon was observed experimentally by adding more of the inducing signal.Plant growth is frequently limited by the essential nutrients nitrogen and phosphorus. Several plant species have established symbiotic relationships with microorganisms to overcome such limitations. In addition to the symbiotic relationship with arbuscular mycorrhizal fungi that many plants establish in order to secure their uptake of water, phosphorus, and other nutrients (Harrison, 2005; Parniske, 2008), legumes have developed interactions with bacteria called rhizobia, resulting in the fixation of atmospheric nitrogen within the plant root (Lhuissier et al., 2001; Gage, 2004; Riely et al., 2006).Root symbioses initiate with signal exchanges in the soil. Plant signals are perceived by the symbionts, triggering the successive release of microbial signals. For rhizobia, the signal molecules are lipochitooligosaccharides termed Nod factors (Dénarié et al., 1996), and the release of lipochitooligosaccharides has also been found in the fungal interaction (Maillet et al., 2011). Upon receiving diffusible signals from the microsymbionts, the plant roots initiate developmental programs that lead to infection by rhizobia or arbuscular mycorrhizal fungi. Both programs employ the same signaling pathway with several components being common to both mycorrhizal and rhizobial interactions (Kistner and Parniske, 2002; Lima et al., 2009). In particular, both the symbioses show characteristic perinuclear and nucleoplasmic localized calcium (Ca2+) oscillations, so-called Ca2+ spiking (Oldroyd and Downie, 2006; Sieberer et al., 2009). It has been suggested that Ca2+ is released from an internal store, most likely the nuclear lumen and associated endoplasmic reticulum (ER; Matzke et al., 2009), with targeted release in the nuclear region (Capoen et al., 2011).Genetic screens in the model legume Medicago truncatula have identified several genes that are required for the plant in the establishment of both symbioses. Two of these, Doesn’t Make Infections1 (DMI1) and DMI2, are essential for the induction of the Ca2+ oscillations, yet the precise roles and mechanisms of these components remain to be determined. DMI2 codes for a plasma membrane receptor-like kinase (Endre et al., 2002; Stracke et al., 2002) that is required to facilitate further signal transduction in the cell (Bersoult et al., 2005). DMI1 is a cation channel located preferentially on the inner nuclear envelope (Ané et al., 2004; Edwards et al., 2007; Riely et al., 2007; Charpentier et al., 2008; Capoen et al., 2011; Venkateshwaran et al., 2012). DMI3 encodes a calcium calmodulin-dependent protein kinase that interacts with downstream components and is thought to be the decoder of the Ca2+ oscillations (Lévy et al., 2004; Mitra et al., 2004; Hayashi et al., 2010). Additional genes coding for three nucleoporins called NUP85, NUP133, and NENA are also required for Ca2+ oscillations (Kanamori et al., 2006; Saito et al., 2007; Groth et al., 2010). The nuclear pore might be involved in trafficking secondary signals and/or ion channels to the inner nuclear membrane. These shared signaling components are collectively referred to as the common Sym pathway.DMI1 plays a key role in the production of Ca2+ oscillations, but its exact mechanism is still unknown. Orthologs of DMI1 have been found; in Lotus japonicus, they are called CASTOR and POLLUX (Charpentier et al., 2008), and in pea (Pisum sativum), SYM8 (Edwards et al., 2007). CASTOR and POLLUX, as well as calcium calmodulin-dependent protein kinase, are highly conserved both in legumes and nonlegumes (Banba et al., 2008; Chen et al., 2009). This highlights the essential role of the Ca2+ oscillations in mycorrhizal signaling.DMI1 is not the channel responsible for the release of Ca2+ (Charpentier et al., 2008; Parniske, 2008; Venkateshwaran et al., 2012) but probably influences the activity of Ca2+ channels. This is similar to how some K⁺ channels act in other plants and yeast (Peiter et al., 2007). Indeed, DMI1 is possibly a K⁺-permeable channel, based on the observation that POLLUX complements K⁺ transport in yeast (Charpentier et al., 2008). In symbiosis, the mode of action of DMI1 could be to allow cations into the nuclear envelope and in that way counterbalance the transmembrane charge that would occur following the release of Ca2+ into the nucleoplasm and cytoplasm. The cation channel could thus change the electrical potential across the nuclear membranes, affecting the opening of the voltage-activated Ca2+ channels (Edwards et al., 2007). This hypothesis is supported by a study reporting a membrane potential over the nuclear envelope in plants (Matzke and Matzke, 1986).Pharmacological evidence and the characteristics of the Ca2+ oscillations supports the involvement of Ca2+ pumps and Ca2+ channels (Engstrom et al., 2002). The pumps are needed to resequester Ca2+ after each release event, actively transporting Ca2+ against the concentration gradient using ATP. A recent study found a SERCA-type Ca2+ ATPase, MCA8, that is located on the inner and outer nuclear envelope of M. truncatula and is required for the symbiotic Ca2+ oscillations (Capoen et al., 2011). Such SERCA pumps are widely distributed on plant membranes, and the variation in their structure points to them being differentially regulated (Sze et al., 2000).Ca2+ channels release Ca2+ from the store, and the mechanism of activating these Ca2+ channels has been hypothesized to be voltage gated (Edwards et al., 2007; Oldroyd and Downie, 2008), but this remains to be verified experimentally. After release of Ca2+ into the cytosol and nucleoplasm, buffers quickly bind to and remove these free ions due to their toxicity to the cell (Sanders et al., 2002). Buffers, i.e. molecules that can bind Ca2+, may play an important role in determining the nonlinear behavior of the oscillatory system for Ca2+ signaling (Falcke, 2004). As numerous Ca2+ buffers are present in cells, it is important to take their contribution into account. Such buffers can also include experimentally introduced dyes and Ca2+ chelators.In Capoen et al. (2011), we investigated the establishment and transmission of spatial waves across the nuclear envelope and demonstrated that the key components for Ca2+ spiking reside on the inner and outer surface of the nuclear membrane. The computational framework we employed for this analysis makes a number of approximations in order to provide the computational efficiency required to perform spatiotemporal simulations. Here, a main focus is to understand the effect of buffers on the Ca2+ oscillations.In this article, we propose a mathematical model based on three key proteins; a Ca2+ ATPase, a voltage-gated Ca2+ channel, and the cation channel DMI1. The model reproduces the symbiotic Ca2+ oscillations, and we further demonstrate that Ca2+-binding proteins can explain initiation, termination, and experimentally observed variation in oscillation patterns. Furthermore, the model predicts that increases in buffering capacity can cause a period of rapid oscillations, and these were observed experimentally.     

Involvement of Inositol 1,4,5-Trisphosphate-Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV-1 Protein Tat

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.     

Endogenous Dopamine Maintains Synchronous Oscillation of Intracellular Calcium in Primary Cultured-Mouse Midbrain Neurons

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.     

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWC^OFF and AWC^ON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWC^ON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWC^ON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWC^ON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.     

A Gradient of Endogenous Calcium Forms in Mucilage of Graviresponding Roots of Zea mays

Agar blocks that contacted the upper sides of tips of horizontally-orientedroots of Zea mays contain significantly less calcium (Ca) thanblocks that contacted the lower sides of such roots. This gravity-inducedgradient of Ca forms prior to the onset of gravicurvature, anddoes not form across tips of vertically-oriented roots or rootsof agravitropic mutants. These results indicate that (1) Cacan be collected from mucilage of graviresponding roots, (2)gravity induces a downward movement of endogenous Ca in mucilageoverlying the root tip, (3) this gravity-induced gradient ofCa does not form across tips of agravitropic roots, and (4)formation of a Ca gradient is not a consequence of gravicurvature.These results are consistent with gravity-induced movement ofCa being a trigger for subsequent redistribution of growth effectors(e.g. auxin) that induce differential growth and gravicurvature. Atomic absorption, calcium, corn, gravitropism (root), Zea mays