Mitochondrial Participation in the Intracellular Ca2+ Network

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca²⁺ uptake and extrusion modulate free cytosolic [Ca²⁺] (Ca_c) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca²⁺ entry, and to monitor Ca²⁺-activated currents that report near-surface [Ca²⁺]. We show that rapid recovery from elevations of Ca_c requires both the mitochondrial Ca²⁺ uniporter and the mitochondrial energization that drives Ca²⁺ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca²⁺] (Ca_m). The indicated resting Ca_m of 100–200 nM is similar to the resting Ca_c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca_m and Ca_c at high temporal resolution shows that, although Ca_m increases less than Ca_c, mitochondrial sequestration of Ca²⁺ is fast and has high capacity. We find that mitochondrial Ca²⁺ uptake limits the rise and underlies the rapid decay of Ca_c excursions produced by Ca²⁺ entry or by mobilization of reticular stores. We also find that subsequent export of Ca²⁺ from mitochondria, seen as declining Ca_m, prolongs complete Ca_c recovery and that suppressing export of Ca²⁺, by inhibition of the mitochondrial Na⁺/ Ca²⁺ exchanger, reversibly hastens final recovery of Ca_c. We conclude that mitochondria are active participants in cellular Ca²⁺ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca²⁺.     

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.     

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.     

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.     

A Cytosolic Serine Endopeptidase from Trypanosoma cruzi Is Required for the Generation of Ca2+ Signaling in Mammalian Cells

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca²⁺-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca²⁺ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca²⁺-signaling activity for mammalian cells.     

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.     

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).     

Regulation of CAX1, an Arabidopsis Ca2+/H+ Antiporter. Identification of an N-Terminal Autoinhibitory Domain

Regulation of Ca(2+) transport determines the duration of a Ca(2+) signal, and hence, the nature of the biological response. Ca(2+)/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca(2+) levels. Analysis of a full-length CAX1 clone suggested that the CAX1 open reading frame contains an additional 36 amino acids at the N terminus that were not found in the original clone identified by suppression of yeast (Saccharomyces cerevisiae) vacuolar Ca(2+) transport mutants. The long CAX1 (lCAX1) could not suppress the yeast Ca(2+) transport defects despite localization to the yeast vacuole. Calmodulin could not stimulate lCAX1 Ca(2+)/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca(2+)/H+ transport. Sequence analysis suggests that a 36-amino acid N-terminal regulatory domain may be present in all Arabidopsis CAX-like genes. Together, these results suggest a structural feature involved in regulation of Ca(2+)/H+ antiport.     

Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ~40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (~5–10 s) rise in cytosolic Ca²⁺ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ~3 mM Ca²⁺ (SW is ~10 mM Ca²⁺). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca²⁺-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca²⁺ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

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.     

Defective Signaling in a Subpopulation of CD4+ T Cells in the Absence of Ca2+/Calmodulin-Dependent Protein Kinase IV

Ca(2+)/calmodulin-dependent protein kinase IV-deficient (CaMKIV(-/-)) mice have been used to investigate the role of this enzyme in CD4(+) T cells. We identify a functional defect in a subpopulation of CD4(+) T cells, characterized by a cell surface marker profile usually found on memory phenotype CD4(+) T cells. Upon T-cell receptor engagement, the mutant cells produce diminished levels of interleukin-2 (IL-2), IL-4, and gamma interferon protein and mRNA. The defect is secondary to an inability to phosphorylate CREB and to induce CREB-dependent immediate-early genes, including c-jun, fosB, fra2, and junB, which are required for cytokine gene induction. In contrast, stimulated naive CD4(+) T cells from CaMKIV(-/-) mice show normal CREB phosphorylation, induction of immediate-early genes, and cytokine production. Thus, in addition to defining an important signaling role for CaMKIV in a subpopulation of T cells, we identify differential signaling requirements for cytokine production between naive T cells and T cells that express cell surface markers characteristic of the memory phenotype.     

Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca²⁺-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca²⁺/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca²⁺-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.     

Intermolecular Failure of L-type Ca2+ Channel and Ryanodine Receptor Signaling in Hypertrophy

Pressure overload–induced hypertrophy is a key step leading to heart failure. The Ca²⁺-induced Ca²⁺ release (CICR) process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca²⁺ channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of “intermolecular failure.” Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca²⁺ release, visualized as “Ca²⁺ spikes,” became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca²⁺ transients in CHT. These data suggested that, within a certain limit, termed the “stability margin,” mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of “hidden” intermolecular failure in CHT has important clinical implications.