Increase in calcium content and Ca2+-ATPase activity in the brain of fasted rats: Comparison with different ages

The effect of fasting on calcium content and Ca²⁺-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of the brain homogenate from young and elderly rats were significantly increased by overnight–fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca²⁺-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca²⁺-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration–induced increase in brain calcium content was greater in elderly r crease in Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca²⁺-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting–induced increase in brain calcium content is involved in Ca²⁺-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy–dependent mechanism in brain calcium accumulation.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Inhibition by Orthovanadate of ATP-Dependent Ca2+ Transport in Microsomes Isolated from Rat Liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5′-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca²⁺ transport activity (half-maximal inhibition at ～10 μM vanadate, corresponding to ～12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca²⁺ transport is noncompetitive with respect to added Ca²⁺ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca²⁺ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Inhibitors of cell division and protoplasmic streaming fail to cause a detectable effect on intracellular calcium levels in stamen-hair cells of Tradescantia virginiana L.

The herbicides amiprophos-methyl (APM) and oryzalin disrupt mitosis and cytokinesis in plant cells by causing the depolymerization of microtubules. These drugs have also been shown to affect calcium sequestration by mitochondria. Controversy thus exists as to whether microtubule depolymerization occurs as a result of direct interaction between the drug and tubulin, or because of elevated intracellular calcium levels resulting from drug interference with calcium regulation. In order to clarify this issue we have directly measured the effect of these herbicides and other cell-motility-altering drugs on intracellular calcium levels in stamen-hair cells of Tradescantia. The results indicate that low levels (1–3 M) of APM and oryzalin can act within 3–7 min causing disorganization of mitosis. Studies using the calcium indicator indo-1 injected into stamen-hair cells to monitor internal levels of calcium, show that at drug concentrations where inhibitory effects on mitosis and-or cytokinesis are clearly seen, APM, oryzalin, isopropyl-N-phenyl carbamate, caffeine and cytochalasin D produce no change in intracellular calcium levels. Furthermore, except for cytochalasin D, these drugs do not inhibit cytoplasmic streaming, a calcium-sensitive process. We conclude that the mode of action of these drugs on the cytoskeleton is independent of an effect on intracellular calcium.Abbreviations and Symbols APM amiprophos-methyl - [Ca²⁺]_i free intracellular calcium ion concentration - CD cytochalasin D - DMSO dimethylsulfoxide - IPC isopropyl N-phenylcarbamate - MT(s) microtubule(s) To whom correspondence should be addressedWe thank Dr. L.C. Morejohn, University of Texas, Austin, for encouraging us to perform this study and for his gift of amiprophosmethyl and oryzalin. We also thank our colleagues at the University of Massachusetts for many helpful discussions. This work has been supported by grants from the U.S. Department of Agriculture (88-37261-3727) and the National Science Foundation (DCB-88-01-01750).     

Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca²⁺-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca²⁺-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent K_m values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (V_max.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E₂ synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca²⁺ stimulated endogenous arachidonate deacylation and prostaglandin E₂ generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca²⁺-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.     

Mitochondrial ryanodine receptors and other mitochondrial Ca permeable channels

Ca²⁺ channels that underlie mitochondrial Ca²⁺ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca²⁺ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca²⁺ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca²⁺ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca²⁺ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca²⁺ channels.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Adenosine triphosphatases of cestodes Bothriocephalus scorpii

Activities and properties of adenosine triphosphatases (ATPases) were studied in mitochondrial and microsomal fractions of cestodes Bothriocephalus scorpii parasitizing in pyloric appendages of the Brandt’s bullhead Myoxocephalus brandti. The highest activity was revealed in the mitochondrial fraction. The mitochondrial and microsomal fractions of B. scorpii had the ATPase activity dependent on the presence of cations Mg²⁺, Mn²⁺, and Ca²⁺. Effects of ions and inhibitors on the B. scorpii ATPase activity with various cations were. Both subcellular fractions were able to hydrolyze, apart from ATP, also GTP, CTP, and UTP.     

Restoration of CA2+-inhibited oxidative phosphorylation in cardiac mitochondria by mitochondrial Ca2+ unloading

Mitochondria, the major source of cellular ATP, display high vulnerability to metabolic stress, in particular to excessive Ca²⁺ loading. Here, we show that Ca²⁺-inhibited mitochondrial ATP generation could be restored through stimulated Ca²⁺ discharge from mitochondrial matrix. This was demonstrated using a Ca²⁺ ionophore or through Na⁺/Ca²⁺ exchange-mediated decrease of mitochondrial Ca²⁺ load. Furthermore, diazoxide, a mitochondrial potassium channel opener, which maintained mitochondrial Ca²⁺ homeostasis, also restored Ca²⁺-inhibited ATP synthesis and preserved the structural integrity of Ca²⁺-challenged mitochondria. Thus, under conditions of excessive mitochondrial Ca²⁺ overload targeting mitochondrial Ca²⁺ transport pathways restores oxidative phosphorylation required for vital cellular processes. This study, therefore, identifies an effective strategy capable to rescue Ca²⁺-disrupted mitochondrial energetics.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca²⁺ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca²⁺ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca²⁺ transport and PTP opening are largely dependent on electron transport and energy coupling.     

TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria

Mitochondrial Ca²⁺ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca²⁺ content and cytosolic Ca²⁺ homeostasis strictly depend on Ca²⁺ transporters. In recent decades, the major players responsible for mitochondrial Ca²⁺ uptake and release have been identified, except the mitochondrial Ca²⁺/H⁺ exchanger (CHE). Originally identified as the mitochondrial K⁺/H⁺ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell‐based and cell‐free biochemical assays demonstrate the absence or greatly reduced Na⁺‐independent mitochondrial Ca²⁺ release in TMBIM5 knockout or pH‐sensing site mutants, respectively, and pH‐dependent Ca²⁺ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long‐sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca²⁺ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca²⁺ exchange.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

The effect of Ca²⁺ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca²⁺ in the range of 0.1–0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca²⁺ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fractions, incubated in the presence of initial Ca²⁺ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.Pregnenolone production from endogenous substrates in mitochondrial fractions isolated in 0.25 M sucrose from control and luteinizing hormone treated testes incubated in the absence of added succinate and Ca²⁺, was maintained during 10–20 min.After longer incubation times no further steroid synthesis took place. Addition of 0.5 mM Ca²⁺ to the incubation medium at time zero slightly stimulated initial pregnenolone production rates in control mitochondrial fractions, but had no effect during prolonged incubations. Addition of 0.5 mM Ca²⁺ to mitochondrial fractions isolated from luteinizing hormone treated glands showed no effect either on initial production rate or during prolonged incubations.Pregnenolone production rates were maintained during 90 min in the presence of 20 mM succinate in the incubation medium. Under such conditions production rates during the first 20 min in mitochondrial fractions obtained from luteinizing hormone treated glands were approx. 3 times higher than in relevant control samples. Addition of 0.5 mM Ca²⁺ to the incubation medium containing 20 mM succinate markedly stimulated initial pregnenolone production rates in control mitochondrial fractions, but gave only a small stimulation of succinate-supported production rates in luteinizing hormone treated testicular mitochondrial fractions. These results indicate that Ca²⁺ in vitro can mimic the trophic effect of luteinizing hormone in vivo on mitochondrial pregnenolone production.Ageing of mitochondrial protein for 60 min at 33°C resulted in a marked increase in pregnenolone production rates in mitochondrial fractions obtained from control testes. The same treatement hardly influenced production rates in mitochondrial fractions isolated from luteinizing hormone treated testes. Ageing may have an effect on the ultrastructure of freshly prepared mitochondria, causing a change in the amount of cholesterol readily available for the enzyme complex.The gluco- and mucoprotein specific agent Ruthenium red (50–2000 ng/ml) did not inhibit pregnenolone production in either control or hormone treated testicular mitochondrial fractions, incubated in the absence of added Ca²⁺. the presence of 200–2000 ng Ruthenium red per ml incubation mixture.The present results have been discussed in relation to the possible involvement of Ca²⁺ in the molecular mechanism of short-term action of luteinizing hormone on testicular androgen production.     

MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots

Ca²⁺ signaling is central to plant development and acclimation. While Ca²⁺-responsive proteins have been investigated intensely in plants, only a few Ca²⁺-permeable channels have been identified, and our understanding of how intracellular Ca²⁺ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca²⁺ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca²⁺ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca²⁺ transport in planta and that this mechanism is the major route for fast Ca²⁺ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca²⁺ imaging in living root tips and the stimulation of Ca²⁺ transients of different amplitudes, we demonstrated that mitochondrial Ca²⁺ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca²⁺ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca²⁺ entry route in planta and link mitochondrial Ca²⁺ transport with phytohormone signaling.

Monitoring of subcellular Ca²⁺ dynamics in living Arabidopsis roots reveals that MCU proteins provide the dominant mitochondrial Ca²⁺ uptake mechanism in vivo.     

The contribution of mitochondrial calcium ion exchange to relaxation of tension in cardiac muscle

The possible contribution of mitochondrial Ca²⁺ accumulation and release to contractile phenomena has been investigated. Two intracellular fractions of Ca²⁺ sequestration can be identified in cardiac myocytes, one ascribed to mitochondria. Two modes of Ca²⁺ transport exist within the mitochondrial fraction, one dependent upon mitochondrial respiration and the other upon extramitochondrial [Na⁺]. Experiments with trabeculae show that under appropriate conditions, the rate of relaxation and the amount of tension developed is dependent on these two modes of Ca²⁺ transport. A model is presented quantifying the contribution of the mitochondria to relaxation.