Calcium uptake mechanisms of mitochondria

The ability of mitochondria to capture Ca²⁺ ions has important functional implications for cells, because mitochondria shape cellular Ca²⁺ signals by acting as a Ca²⁺ buffer and respond to Ca²⁺ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca²⁺ channel known as the uniporter drives the rapid and massive entry of Ca²⁺ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca²⁺ concentrations that are only reached transiently in cells, near Ca²⁺ release channels. Mitochondria can also take up Ca²⁺ at low, nanomolar concentrations, but this high affinity mode of Ca²⁺ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca²⁺/H⁺ antiporter that drives the uptake of Ca²⁺ into mitochondria at nanomolar cytosolic Ca²⁺ concentrations. In this article, we will review the properties of the Ca²⁺ import systems of mitochondria and discuss how Ca²⁺ uptake via an electrogenic 1:1 Ca²⁺/H⁺ antiport challenges our current thinking of the mitochondrial Ca²⁺ uptake mechanism.     

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca²⁺ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca²⁺ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca²⁺ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca²⁺ that will enable it to act as a Ca²⁺ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca²⁺] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca²⁺ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca²⁺ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.     

Extracellular calcium is involved in stomatal movement through the regulation of water channels in broad bean

Involvement of extracellular Ca²⁺ in stomatal movement through the regulation of water channels was investigated in broad bean (Vicia faba L.). Leaf peels were first incubated to open stomata, and then transferred to buffers in the presence of different CaCl₂ concentrations. Stomatal status was observed under magnification and stomatal aperture (pore width/length) was measured. Stomatal closure was significantly induced and aperture oscillation occurred at lower extracellular concentrations of calcium ([Ca²⁺]_ext), while at higher concentrations, no significant change in stomatal aperture was observed, which was similar to the response recorded with HgCl₂. Lower [Ca²⁺]_ext-induced stomatal closure could be reversed using depolarizing buffer. It is suggested that lower [Ca²⁺]_ext regulates water channels through an indirect way and at higher concentrations, extracellular Ca²⁺ is involved in regulating stomatal aperture by directly influencing water channels to retard aperture change.     

Unique isoform-specific properties of calsequestrin in the heart and skeletal muscle

Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca²⁺ concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca²⁺ binding to CSQ2 is 50% lower than to CSQ1. Only ～30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with ～70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca²⁺ release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca²⁺ and allow repeated Ca²⁺ release and graded activation with increased stimulation frequency.     

Crosstalk between insulin receptor and G protein-coupled receptor signaling systems leads to Ca oscillations in pancreatic cancer PANC-1 cells

We examined crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways in individual human pancreatic cancer PANC-1 cells. Treatment of cells with insulin (10 ng/ml) for 5 min markedly enhanced the proportion of cells that display an increase in intracellular [Ca²⁺] induced by picomolar concentrations of the GPCR agonist neurotensin. Interestingly, insulin increased the proportion of a subpopulation of cells that exhibit intracellular [Ca²⁺] oscillations in response to neurotensin at concentrations as low as 50-200 pM. Insulin enhanced GPCR-induced Ca²⁺ signaling in a time- and dose-dependent manner; a marked potentiation was obtained after an exposure to a concentration of 10 ng/ml for 5 min. Treatment with the mTORC1 inhibitor rapamycin abrogated the increase in GPCR-induced [Ca²⁺]_i oscillations produced by insulin. Our results identify a novel aspect in the crosstalk between insulin receptor and GPCR signaling systems in pancreatic cancer cells, namely that insulin increases the number of [Ca²⁺]_i oscillating cells induced by physiological concentrations of GPCR agonists through an mTORC1-dependent pathway.     

Ca-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. , 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 (Ca²⁺/monomer). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Redox regulation of calcium ion channels: chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease.Changes in the intracellular free Ca²⁺ concentration are key triggers for diverse cellular functions. Ca²⁺ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca²⁺ permeable ion channels such as voltage-gated (CaV) Ca²⁺ channels, transient receptor potential (TRP) channels and Orai channels.     

Calcium Inhibits Dihydropyridine-Stimulated Increases in Opening and Unitary Conductance of a Plant Ca<Superscript>2+</Superscript> Channel

We have previously characterized the “RCA” channel (root Ca²⁺ channel), a voltage-dependent, Ca²⁺-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers. Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202–791). However, the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca²⁺, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca²⁺ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202–791 or nifedipine caused an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive interaction between Ca²⁺ and DHPs at one of the Ca²⁺-binding sites involved in Ca²⁺ permeation and that binding of a DHP to one of the Ca²⁺-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant Ca²⁺-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between plant and animal Ca²⁺-permeable channels.     

Regulation of the gating of the sheep cardiac sarcoplasmic reticulum ca2+-release channel by luminal Ca2+

We investigated the effects of changes in luminal [Ca²⁺] on the gating of native andpurified sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels reconstituted intoplanar phospholipid bilayers. The open probability (P _o )of channels activated solely by cytosolic Ca²⁺ was greater at positive than negative holding potentials. Channels activatedsolely by 10 m cytosolic Ca²⁺ exhibited no change in steady-stateP _o or in the relationship betweenP _o and voltage when the luminal[Ca²⁺] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrationsof cytosolic Ca²⁺, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolicCa²⁺-independent activation of the channel by sulmazole requires luminal Ca²⁺. In the presence ofsulmazole, at picomolar luminal [Ca²⁺] the channel remains completely closed. Increasing the luminal [Ca²⁺]to millimolar levels markedly increases the P _o via an increase in theduration of open events. The P _o and duration of the sulmazole-activated, luminalCa²⁺-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca²⁺, theP _o and duration of sulmazole-activated openings are greater atnegative voltages. However, at millimolar luminal [Ca²⁺], long openings are also observed at positive voltages and theP _o appears to be similar at positive and negative voltages. Our findings indicate thatthe regulation of channel gating by luminal Ca²⁺ depends on the mechanism of channel activation.We would like to thank Dr Allan Lindsay for the preparation of the purified SR Ca²⁺-release channels. This work was supported by the British Heart Foundation.     

Growth factor-induced proliferation of human fibroblasts in serum-free culture depends on cell density and extracellular calcium concentration

Human neonatal skin fibroblasts plated sparsely in MCDB 105 traversed a complete cell cycle in the absense of serum or serum-derived proteins. Addition of pure PDGF did not significantly increase entrance into S phase as revealed by ³H-thymidine labeling index or clonal growth on palladium islands. In subphysiologic Ca²⁺ concentrations or in the presence of a calmodulin inhibitor, W7, proliferation in the absence of growth factors ceased and PDGF became mitogenic. In contrast, confluent fibroblast cultures were stimulated by PDGF in physiologic Ca²⁺ concentrations. This was also the case with sparse adult skin fibroblast cultures while a fetal strain entered S in the absence of PDGF even in low extracellular Ca²⁺ concentrations. EGF gave similar results as PDGF in all experiments performed. This proposes a similar role for the two growth factors in the cell cycle. However, a difference in the mechanisms of action of PDGF and EGF is indicated by the fact that PDGF and EGF were additive at optimal concentrations when maximal growth response by a single growth factor was restricted by a subphysiologic extracellular Ca²⁺ concentration.     

Cell adhesion and intracellular calcium signaling in neurons

Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca²⁺ levels play a prominent role. Ca²⁺ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca²⁺ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca²⁺ sources and channels involved in intracellular Ca²⁺ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca²⁺ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca²⁺ stores in neurons are discussed.     

Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating

Polycystin-2 (PC2) is a Ca²⁺-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca²⁺. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca²⁺-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca²⁺ remains unknown. We propose that the PC2 EF-hand is a Ca²⁺ sensor required for channel gating. Consistent with this, Ca²⁺ binding causes a dramatic decrease in the radius of gyration (R_g) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca²⁺ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D_max) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca²⁺ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca²⁺ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca²⁺ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca²⁺-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.     

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor,ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn²⁺ in accelerating wound healing is established, yet, the signaling pathways linking Zn²⁺ to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn²⁺ induces a metabotropic Ca²⁺ response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn²⁺-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn²⁺ and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn²⁺ was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn²⁺ probe, ZnAF-2. Chelation of Zn²⁺ and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca²⁺ signaling. The signaling triggered by Zn²⁺, via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn²⁺, which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. $\text{256}$, 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 ($\text{Ca}{}^{\mathrm{2+}}\text{monomer}$). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling"

Abstract Interaction between a T cell and an antigen‐presenting cell leads to the rapid formation of an immunological synapse allowing antigen detection by the T cell and the development of an immune response. Antigen detection triggers various cellular responses including a modest but sustained T cell Ca²⁺ increase. In this review are discussed a series of related questions. What are the various molecular events by which a T cell Ca²⁺ response can be triggered in the immunological synapse by a very small amount of antigen ? How is Ca²⁺ released from intracellular stores and how can these stores remain empty for hours ? Through which channels does Ca²⁺ influx takes place, and how is Ca²⁺ influx coupled to Ca²⁺ release from intracellular stores ? What are the main immediate and indirect cellular targets of the Ca²⁺ increase ?     

Simultaneous addition of EGF prolongs the increase in cytosolic free calcium seen in response to bradykinin in NRK-49F cells

The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca²⁺]_i,) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca²⁺]_i, which generally decayed to near basal [Ca²⁺]_i within 3 minutes. The initial rise in [Ca²⁺]_i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca²⁺]_i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca²⁺]_i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca²⁺]_i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca²⁺]_i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca²⁺]_i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.     

Calcium Uptake and Catecholamine Secretion by Cultured Bovine Adrenal Medulla Cells

Abstract: The uptake of ⁴⁵Ca²⁺ and secretion of catecholamines by primary cultures of adrenal medulla cells were studied. Nicotine, veratridine, potassium, and Ionomycin stimulate both the accumulation of ⁴⁵Ca²⁺ and the secretion of catecholamines. Nicotinic antagonists block ⁴⁵Ca²⁺ uptake induced by nicotine, tetrodotoxin blocks ⁴⁵Ca²⁺ uptake induced by veratridine, and D600 blocks uptake induced by K⁺, nicotine, and veratridine, but not ⁴⁵Ca²⁺ uptake or secretion induced by Ionomycin. The EC₅₀ for nicotine is 3 μm for catecholamine secretion and 10 μm for ⁴⁵Ca²⁺ uptake, while the EC₅₀S for veratridinestimulated uptake and secretion are approximately the same (75 μm ). Kinetic studies show that the uptake of Ca²⁺ is rapid and appears to precede the secretion of catecholamines, and that the rate of uptake declines rapidly. The uptake of ⁴⁵Ca²⁺ and secretion of catecholamines stimulated by veratridine and 50 mm -K⁺ show saturation kinetics with respect to external calcium concentrations at about 2 mm . On the other hand, the uptake of ⁴⁵ Ca²⁺ stimulated by nicotine does not become saturated at external calcium concentrations of 10 mm although the secretion of catecholamines reaches a maximum at external calcium concentrations of 2 mm . The data suggest that depolarizing agents such as veratridine and 50 mm -K⁺ stimulate ⁴⁵Ca²⁺ entry through voltage-sensitive calcium channels, while nicotinic agonists stimulate calcium entry through the acetylcholine receptor ion channels as well as through voltage-sensitive calcium channels.