Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca²⁺] ([Ca²⁺]_i) and subsequent activation of K_Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/Principal Findings

We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca²⁺- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca²⁺]_i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca²⁺]_i in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/Significance

The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca²⁺-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca²⁺ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.     

Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background

Ca²⁺ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) in the prefusion phase, the occurrence and significance of Ca²⁺ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca²⁺- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca²⁺]_c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t_1/2 of decay = 3.2 s) rise of localized [Ca²⁺]_c originating at the site of lamellar body fusion. [Ca²⁺]_c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca²⁺ from, or addition of Ni²⁺ to the extracellular solution, strongly inhibited these [Ca²⁺]_c transients, whereas Ca²⁺ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca²⁺]_c. Both effects were reduced by the non-specific Ca²⁺ channel blocker {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365.

Conclusions/Significance

Fusion-activated Ca²⁺ entry (FACE) is a new mechanism that leads to [Ca²⁺]_c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca²⁺ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.     

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.     

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium

Introduction

Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).

Methods

Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca²⁺]_i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca²⁺]_i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.

Results

Acidic pH increased [Ca²⁺]_i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca²⁺]_i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca²⁺]_i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca²⁺]_i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.

Conclusions

Decreased pH activates ASIC3 resulting in increased [Ca²⁺]_i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca²⁺]_i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.     

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca²⁺-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca²⁺ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca²⁺ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca²⁺ is quenched to or below resting intracellular Ca²⁺ concentration ([Ca²⁺]_e [Ca²⁺]_i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca²⁺]_e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca²⁺]_i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca²⁺ mobilization from stores (providing close to threshold Ca²⁺ levels) and Ca²⁺ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca²⁺ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.     

Loss of Pde6 reduces cell body Ca2+ transients within photoreceptors

Modulation of Ca²⁺ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca²⁺ indicators to define changes in perikaryal Ca²⁺ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca²⁺]_i levels within the cell body. Arguing against Ca²⁺ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca²⁺ fluxes revealed that degeneration of pde6c^w59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca²⁺]_i. Analysis of [Ca²⁺]_i in dissociated Pde6β^rd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca²⁺ elevations (‘flashes'') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca²⁺ fluxes diminished. This study thus provides insight into Ca²⁺ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca²⁺]_i that occurs in normal cones.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca²⁺ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca²⁺ responses in ipRGCs, the light-evoked rise in intracellular Ca²⁺ ([Ca²⁺]_i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca²⁺]_i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca²⁺]_i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca²⁺]_i in isolated ipRGCs is almost entirely due to Ca²⁺ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca²⁺]_i in ipRGCs by blocking L-type voltage-gated Ca²⁺ channels. The ability of carbenoxolone to block evoked Ca²⁺ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca²⁺]_i is the output being measured.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

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.     

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell''s diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.

Methodology/Principal Findings

The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.

Conclusions/Significance

The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.     

Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells

Two recombinant aequorin isoforms with different Ca²⁺ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca²⁺ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca²⁺]_er or [Sr²⁺]_er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca²⁺] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca²⁺ concentration, the [Ca²⁺]_er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca²⁺]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr²⁺ was used as a Ca²⁺ surrogate), the bulk of the organelle was shown to accumulate free [cation²⁺]_er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca²⁺], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca²⁺ or Sr²⁺, is presented. Moreover, a few other key problems concerning the ER Ca²⁺ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP₃ vary depending on their initial [Ca²⁺]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca²⁺] the cation is simultaneously accumulated; (b) stimulation of Ca²⁺ release by receptor-generated InsP₃ is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation²⁺]_er of the InsP₃ receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca²⁺], observed when the cells are incubated in Ca²⁺-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).     

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.     

Capacitative Ca2+ Entry Is Closely Linked to the Filling State of Internal Ca2+ Stores: A Study Using Simultaneous Measurements of ICRAC and Intraluminal [Ca2+]

I_CRAC (the best characterized Ca²⁺ current activated by store depletion) was monitored concurrently for the first time with [Ca²⁺] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca²⁺] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca²⁺ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca²⁺] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca²⁺ pumps or release channels. Using these new tools, we demonstrate here that Ca²⁺ release–activated Ca²⁺ current (I_CRAC) is activated (a) solely by reduction of free [Ca²⁺] within the ER and (b) by any measurable decrease in [Ca²⁺]_ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.     

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca²⁺ spikes” (i.e., [Ca²⁺]_c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca²⁺ spikes are related to the slow circadian rhythms.

Methodology/Principal Findings

We addressed this issue based on a Ca²⁺ indicator dye (fluo-4) and a protein Ca²⁺ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca²⁺ spikes in 18% of rat SCN cells in acute brain slices, but the Ca²⁺ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca²⁺ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca²⁺ spikes was increased to 13∼14%.

Conclusions/Significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca²⁺ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca²⁺ spiking activity is caused by the Ca²⁺ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca²⁺]_c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca²⁺ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca²⁺ spikes in the function of SCN.     

The Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca2+ Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H2O2-Induced Apoptosis with Ca2+ Overload

Intracellular calcium concentration ([Ca²⁺]_i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca²⁺]_i and its control mechanisms in process of hydrogen peroxide (H₂O₂)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca²⁺ indicator to detect [Ca²⁺]_i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H₂O₂-induced retinal cell apoptosis occurred at 4 h after H₂O₂ stress and increased in a time-dependent manner, but [Ca²⁺]_i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H₂O₂ stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca²⁺]_i, which appeared only at 0.5 h after βE2 application; c) increased [Ca²⁺]_i under 100 µM H₂O₂ treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca²⁺ stores; d) importantly, the transiently increased [Ca²⁺]_i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca²⁺ channels (L-VGCC), but the increased [Ca²⁺]_i induced by 100 µM H₂O₂ treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H₂O₂, which was also associated with its transient [Ca²⁺]_i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.     

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.