Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP₃R)-operated Ca²⁺ stores interact as coupled oscillators to produce near-synchronous Ca²⁺ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP₃, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca²⁺ transients and/or intracellular Ca²⁺ waves. Larger near-synchronous Ca²⁺ transients then occurred leading to global synchronous Ca²⁺ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca²⁺ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca²⁺ transients and resultant action potentials, leaving only asynchronous Ca²⁺ transients and local Ca²⁺ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca²⁺ release from IP₃R-operated Ca²⁺ stores, which causes depolarization; 2), L-type Ca²⁺ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca²⁺ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca²⁺ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca²⁺ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca²⁺ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca²⁺ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca²⁺ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release: cytoplasmic and luminal regulation modeled in a tetrameric channel

In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]_L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release.     

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca²⁺ influx via voltage-gated L-type Ca²⁺ channels activates Ca²⁺ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca²⁺]_i transients in hESC-CMs required Ca²⁺ entry through L-type Ca²⁺ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca²⁺ or treatment with diltiazem, an L-type channel inhibitor. Ca²⁺ release from the SR also contributes to the [Ca²⁺]_i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca²⁺ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca²⁺ currents (I _Ca) and corresponding whole-cell [Ca²⁺]_i transients in hESC-CMs and hFVMs, and the amplitude of both I _Ca and the [Ca²⁺]_i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca²⁺]_i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca²⁺ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca²⁺ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca²⁺ release by the I _Ca during EC coupling develops early in human cardiomyocytes.     

Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA

In the present study we describe the analysis of optically recorded whole cell Ca²⁺ transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca²⁺ mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca²⁺-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca²⁺ to the myoplasmic space (Ca²⁺ input flux). The validity of the procedure was confirmed by model simulations using artificial Ca²⁺ input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca²⁺ input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca²⁺ flux carried by the L-type Ca²⁺ channels, indicating that it consists mainly of voltage-controlled Ca²⁺ release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca²⁺ inward current, indicating that Ca²⁺ release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca²⁺ release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α_1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 μM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Identification of PTEN at the ER and MAMs and its regulation of Ca2+ signaling and apoptosis in a protein phosphatase-dependent manner

The tumor suppressor activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10) is thought to be largely attributable to its lipid phosphatase activity. PTEN dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate to directly antagonize the phosphoinositide 3-kinase-Akt pathway and prevent the activating phosphorylation of Akt. PTEN has also other proposed mechanisms of action, including a poorly characterized protein phosphatase activity, protein–protein interactions, as well as emerging functions in different compartment of the cells such as nucleus and mitochondria. We show here that a fraction of PTEN protein localizes to the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), signaling domains involved in calcium (²⁺) transfer from the ER to mitochondria and apoptosis induction. We demonstrate that PTEN silencing impairs ER Ca²⁺ release, lowers cytosolic and mitochondrial Ca²⁺ transients and decreases cellular sensitivity to Ca²⁺-mediated apoptotic stimulation. Specific targeting of PTEN to the ER is sufficient to enhance ER-to-mitochondria Ca²⁺ transfer and sensitivity to apoptosis. PTEN localization at the ER is further increased during Ca²⁺-dependent apoptosis induction. Importantly, PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and this correlates with the reduction in their phosphorylation and increased Ca²⁺ release. We propose that ER-localized PTEN regulates Ca²⁺ release from the ER in a protein phosphatase-dependent manner that counteracts Akt-mediated reduction in Ca²⁺ release via IP3Rs. These findings provide new insights into the mechanisms and the extent of PTEN tumor-suppressive functions, highlighting new potential strategies for therapeutic intervention.     

Peripheral Hot Spots for Local Ca2+ Release after Single Action Potentials in Sympathetic Ganglion Neurons

Ca²⁺ release from the endoplasmic reticulum (ER) contributes to Ca²⁺ transients in frog sympathetic ganglion neurons. Here we use video-rate confocal fluo-4 fluorescence imaging to show that single action potentials reproducibly trigger rapidly rising Ca²⁺ transients at 1–3 local hot spots within the peripheral ER-rich layer in intact neurons in fresh ganglia and in the majority (74%) of cultured neurons. Hot spots were located near the nucleus or the axon hillock region. Other regions exhibited either slower and smaller signals or no response. Ca²⁺ signals spread into the cell at constant velocity across the ER in nonnuclear regions, indicating active propagation, but spread with a (time)^1/2 dependence within the nucleus, consistent with diffusion. 26% of cultured cells exhibited uniform Ca²⁺ signals around the periphery, but hot spots were produced by loading the cytosol with EGTA or by bathing such cells in low-Ca²⁺ Ringer's solution. Peripheral hot spots for Ca²⁺ release within the perinuclear and axon hillock regions provide a mechanism for preferential initiation of nuclear and axonal Ca²⁺ signals by single action potentials in sympathetic ganglion neurons.     

Causes of Abnormal Ca2+ Transients in Guinea Pig Pathophysiological Ventricular Muscle Revealed by Ca2+ and Action Potential Imaging at Cellular Level

Background

Abnormal Ca²⁺ transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca²⁺ transients can be attributed to abnormal action potential generation or abnormal Ca²⁺ handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca²⁺ and action potential signals at the cellular level in isolated heart tissues.

Methodology/Principal Findings

After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca²⁺ transients. For the failed and alternating Ca²⁺ transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca²⁺ transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na⁺ channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na⁺ channel inactivation.

Conclusions/Significance

Our results indicate that there are multiple patterns for the causes of abnormal Ca²⁺ signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.     

Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Dysregulation of intracellular Ca²⁺ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer''s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ_1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca²⁺ in neurons and promote mitochondrial Ca²⁺ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca²⁺ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca²⁺ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca²⁺ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca²⁺ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Filtering of calcium transients by the endoplasmic reticulum in pancreatic beta-cells

Calcium handling in pancreatic β-cells is important for intracellular signaling, the control of electrical activity, and insulin secretion. The endoplasmic reticulum (ER) is a key organelle involved in the storage and release of intracellular Ca²⁺. Using mathematical modeling, we analyze the filtering properties of the ER and clarify the dual role that it plays as both a Ca²⁺ source and a Ca²⁺ sink. We demonstrate that recent time-dependent data on the free Ca²⁺ concentration in pancreatic islets and β-cell clusters can be explained with a model that uses a passive ER that takes up Ca²⁺ when the cell is depolarized and the cytosolic Ca²⁺ concentration is elevated, and releases Ca²⁺ when the cell is repolarized and the cytosolic Ca²⁺ is at a lower concentration. We find that Ca²⁺-induced Ca²⁺ release is not necessary to explain the data, and indeed the model is inconsistent with the data if Ca²⁺-induced Ca²⁺ release is a dominating factor. Finally, we show that a three-compartment model that includes a subspace compartment between the ER and the plasma membrane provides the best agreement with the experimental Ca²⁺ data.     

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca²⁺ entry (SOCE) to Ca²⁺ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn²⁺ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca²⁺ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca²⁺ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca²⁺ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca²⁺ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca²⁺ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca²⁺ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca²⁺ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background

Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca²⁺ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca²⁺ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.

Methodology/Principal Findings

By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca²⁺ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca²⁺; iii) GDPβS has little effect on the amplitude of the Ca²⁺ peak, but significantly expedites the initial Ca²⁺ waves induced by InsP₃ photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca²⁺ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca²⁺ signaling.

Conclusions/Significance

Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca²⁺ release.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Background

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca²⁺] ([Ca²⁺]_o) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca²⁺]_o sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.

Methodology/Principal Findings

By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca²⁺]_o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.

Conclusions/Significance

Our experiments identify CaSR as the first component in the [Ca²⁺]_o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca²⁺]_o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca²⁺]_o following its entry via voltage-activated Ca²⁺ channels. CaSR may detects such falls in [Ca²⁺]_o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca²⁺]_o on release probability. CaSR is positioned to detect the dynamic changes of [Ca²⁺]_o and provide presynaptic feedback that will alter brain excitability.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.