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.     

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.     

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.     

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.     

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.     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

A Calcium Signaling Cascade Essential for Myosin Thick Filament Assembly in Xenopus Myocytes

Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca²⁺ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca²⁺ release from the ryanodine receptor (RyR) activated Ca²⁺ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.     

Ca2+ Regulates Reactive Oxygen Species Production and pH during Mechanosensing in Arabidopsis Roots

Mechanical stimulation of plants triggers a cytoplasmic Ca²⁺ increase that is thought to link the touch stimulus to appropriate growth responses. We found that in roots of Arabidopsis thaliana, external and endogenously generated mechanical forces consistently trigger rapid and transient increases in cytosolic Ca²⁺ and that the signatures of these Ca²⁺ transients are stimulus specific. Mechanical stimulation likewise elicited an apoplastic alkalinization and cytoplasmic acidification as well as apoplastic reactive oxygen species (ROS) production. These responses showed the same kinetics as mechanically induced Ca²⁺ transients and could be elicited in the absence of a mechanical stimulus by artificially increasing Ca²⁺ concentrations. Both pH changes and ROS production were inhibited by pretreatment with a Ca²⁺ channel blocker, which also inhibited mechanically induced elevations in cytosolic Ca²⁺. In trichoblasts of the Arabidopsis root hair defective2 mutant, which lacks a functional NADPH oxidase RBOH C, touch stimulation still triggered pH changes but not the local increase in ROS production seen in wild-type plants. Thus, mechanical stimulation likely elicits Ca²⁺-dependent activation of RBOH C, resulting in ROS production to the cell wall. This ROS production appears to be coordinated with intra- and extracellular pH changes through the same mechanically induced cytosolic Ca²⁺ transient.     

Light-induced changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H⁺, Ca²⁺, K⁺, and Cl⁻ fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia faba L.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca²⁺ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H⁺, K⁺, and Cl⁻ occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca²⁺ began within the time resolution of our measurements (5 s). In the absence of H⁺ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO₂ uptake by photosynthesizing tissue, whereas activation of the plasma membrane H⁺ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO₂ diffusion into the leaf.     

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca²⁺ chelator EGTA. Endosome fusion was restored by the addition of Ca²⁺ with an optimum at a free Ca²⁺ concentration of 0.3 × 10⁻⁶ M. Other divalent cations did not substitute for Ca²⁺. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca²⁺. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca²⁺ from the endosomal interior.     

Ca2+ Current and Charge Movements in Skeletal Myotubes Promoted by the β-Subunit of the Dihydropyridine Receptor in the Absence of Ryanodine Receptor Type 1

The β-subunit of the dihydropyridine receptor (DHPR) enhances the Ca²⁺ channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the β-subunit in the expression of L-type Ca²⁺ current and charge movements in RyR1 knock-out (KO), β1 KO, and double β1/RyR1 KO myotubes generated by mating heterozygous β1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various β-isoforms and then whole-cell voltage-clamped for measurements of Ca²⁺ and gating currents. Overexpression of the endogenous skeletal β1a isoform resulted in a low-density Ca²⁺ current either in RyR1 KO (36 ± 9 pS/pF) or in β1/RyR1 KO (34 ± 7 pS/pF) myotubes. However, the heterologous β2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca²⁺ current that was entirely wild-type in density in RyR1 KO (195 ± 16 pS/pF) and was significantly enhanced in double β1/RyR1 KO (115 ± 18 pS/pF) myotubes. Other variants tested from the four β gene families (β1a, β1b, β1c, β3, and β4) were unable to enhance Ca²⁺ current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in β2a-expressing β1a/RyR1 KO myotubes were significantly lower than in β1a-expressing β1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, β2a had a preferential ability to recover Ca²⁺ current, whereas β1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (β2a C3,4S) eliminated the RyR1-independent Ca²⁺ current expression. Furthermore, Ca²⁺ current enhancement was observed with a β2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the β2a variant to the myotube surface activated the DHPR Ca²⁺ current and bypassed the requirement for RyR1. The data suggest that the Ca²⁺ current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the β1a-subunit is critically involved in process.     

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.     

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.

     

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.     

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.     

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.     

Cysteine String Protein Functions Directly in Regulated Exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca²⁺ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca²⁺ signals after depolarization, suggesting that Csp1 has no gross effect on Ca²⁺ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca²⁺ or GTPγS in the absence of Ca²⁺. Because secretion from permeabilized cells is not influenced by Ca²⁺ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Imaging Single Cardiac Ryanodine Receptor Ca2+ Fluxes in Lipid Bilayers

In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca²⁺ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca²⁺ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca²⁺ (25–50 mM) that result in saturating RyR2 Ca²⁺ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca²⁺ currents having amplitudes of 0.25–14 pA. Ca²⁺ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca²⁺ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca²⁺ currents, and for addressing other current research questions is discussed.     

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.