Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Ca(2+)-dependent proteolysis in muscle wasting

Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

Ca(2+)-dependent annexin self-association on membrane surfaces

Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1:1 or 1:3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1:0, 1:1, or 1:3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca(2+)-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levels where the protein may bind two membranes as a monomer.     

Ca2+-dependent nuclear export mediated by calreticulin

We have characterized a pathway for nuclear export of the glucocorticoid receptor (GR) in mammalian cells. This pathway involves the Ca2+ -binding protein calreticulin (CRT), which directly contacts the DNA binding domain (DBD) of GR and facilitates its delivery from the nucleus to the cytoplasm. In the present study, we investigated the role of Ca2+ in CRT-dependent export of GR. We found that removal of Ca2+ from CRT inhibits its capacity to stimulate the nuclear export of GR in digitonin-permeabilized cells and that the inhibition is due to the failure of Ca2+-free CRT to bind the DBD. These effects are reversible, since DBD binding and nuclear export can be restored by Ca2+ addition. Depletion of intracellular Ca2+ inhibits GR export in intact cells under conditions that do not inhibit other nuclear transport pathways, suggesting that there is a Ca2+ requirement for GR export in vivo. We also found that the Ran GTPase is not required for GR export. These data show that the nuclear export pathway used by steroid hormone receptors such as GR is distinct from the Crm1 pathway. We suggest that signaling events that increase Ca2+ could positively regulate CRT and inhibit GR function through nuclear export.     

Ca(2+)-dependent desensitization of insulin secretion by strong potassium depolarization

Depolarization by a high K(+) concentration is a widely used experimental tool to stimulate insulin secretion. The effects occurring after the initial rise in secretion were investigated here. After the initial peak a fast decline occurred, which was followed by a slowly progressive decrease in secretion when a strong K(+) depolarization was used. At 40 mM KCl, but not at lower concentrations, the decrease continued when the glucose concentration was raised from 5 to 10 mM, suggesting an inhibitory effect of the K(+) depolarization. When tolbutamide was added instead of the glucose concentration being raised, a complete inhibition down to prestimulatory values was observed. Equimolar reduction of the NaCl concentration to preserve isoosmolarity enabled an increase in secretion in response to glucose. Unexpectedly, the same was true when the Na(+)-reduced media were made hyperosmolar by choline chloride or mannitol. The insulinotropic effect of tolbutamide was not rescued by the compensatory reduction of NaCl, suggesting a requirement for activated energy metabolism. These inhibitory effects could not be explained by a lack of depolarizing strength or by a diminished free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Rather, the complexation of extracellular Ca(2+) concomitant with the K(+) depolarization markedly diminished [Ca(2+)](i) and attenuated the inhibitory action of 40 mM KCl. This suggests that a strong but not a moderate depolarization by K(+) induces a [Ca(2+)](i)-dependent, slowly progressive desensitization of the secretory machinery. In contrast, the decline immediately following the initial peak of secretion may result from the inactivation of voltage-dependent Ca(2+) channels.     

Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.     

Inability of 50 Hz magnetic fields to regulate PKC- and Ca(2+)-dependent gene expression in Jurkat cells

We have previously reported that the T cell line Jurkat registers the exposure of a sinusoidal extremely low frequency magnetic field at the level of the plasma membrane, resulting in activation of the tyrosine kinase p56(lck), increase in inositol-3-phosphate levels and increase in intracellular calcium concentration within minutes. To elucidate if these events associated with changes in intracellular calcium ion levels were biologically significant, transient transfections of Jurkat cells were performed with calcium-ion dependent reporter constructs. Three different enhancer/promoter constructs were studied coupled to the luciferase reporter gene. The luciferase activity of each construct was measured after treatment of transfected cells to EMF exposure alone, or in combination with ionomycin, phorbol ester or cross-linking anti-CD3 antibodies. There was no indication that the used EMFs could influence any of these reporter constructs.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.