Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Bimodal regulation of Na+–Ca2+ exchanger by β-adrenergic signaling pathway in shark ventricular myocytes

In shark heart, the Na+–Ca2+ exchanger serves as a major pathway for both Ca2+ influx and efflux, as there is only rudimentary sarcoplasmic reticulum in these hearts. The modulation of the exchanger by a β-adrenergic agonist in whole-cell clamped ventricular myocytes was compared with that of the Na+–Ca2+ exchanger blocker KB-R7943. Application of 5 μM isoproterenol and 10 μM KB-R7943 suppressed both the inward and the outward Na+–Ca2+ exchanger current (INa−Ca). The isoproterenol effect was mimicked by 10 μM forskolin. Isoproterenol and forskolin shifted the reversal potential (Erev) of INa−Ca by approximately −23 mV and −30 mV, respectively. An equivalent suppression of outward INa−Ca by KB-R7943 to that by isoproterenol produced a significantly smaller shift in Erev of about −4 mV. The ratio of inward to outward exchanger currents was also significantly larger in isoproterenol- than in control- and KB-R7943-treated myocytes. Our data suggest that the larger ratio of inward to outward exchanger currents as well as the larger shift in Erev with isoproterenol results from the enhanced efficacy of Ca2+ efflux via the exchanger. The protein kinase A-mediated bimodal regulation of the exchanger in parallel with phosphorylation of the Ca2+ channel and enhancement of its current may have evolved to satisfy the evolutionary needs for accelerated contraction and relaxation in hearts of animals with vestigial sarcoplasmic Ca2+ release stores.

Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca2+-activated K+ channel (BK channel) contains Ca2+-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca2+ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45Ca2+-overlay protein blot assay to detect Ca2+ binding. Dslo-C280 exhibits specific binding of 45Ca2+ in comparison with various control proteins and known EF-hand Ca2+-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the “Ca-bowl” motif are changed to Asn, reduces 45Ca2+-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca2+ sensitivity for activation and a shift of ≈+80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca2+ and a reduction in apparent Ca2+ affinity, suggesting the loss of one Ca2+-binding site per monomer. These results demonstrate a functional correlation between Ca2+ binding to a specific region of the BK protein and Ca2+-dependent activation, thus providing a biochemical approach to study this process.

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa1 : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Ultra-low concentrations of naloxone selectively antagonize excitatory effects of morphine on sensory neurons, thereby increasing its antinociceptive potency and attenuating tolerance/dependence during chronic cotreatment.

Ultra-low picomolar concentrations of the opioid antagonists naloxone (NLX) and naltrexone (NTX) have remarkably potent antagonist actions on excitatory opioid receptor functions in mouse dorsal root ganglion (DRG) neurons, whereas higher nanomolar concentrations antagonize excitatory and inhibitory opioid functions. Pretreatment of naive nociceptive types of DRG neurons with picomolar concentrations of either antagonist blocks excitatory prolongation of the Ca(2+)-dependent component of the action potential duration (APD) elicited by picomolar-nanomolar morphine and unmasks inhibitory APD shortening. The present study provides a cellular mechanism to account for previous reports that low doses of NLX and NTX paradoxically enhance, instead of attenuate, the analgesic effects of morphine and other opioid agonists. Furthermore, chronic cotreatment of DRG neurons with micromolar morphine plus picomolar NLX or NTX prevents the development of (i) tolerance to the inhibitory APD-shortening effects of high concentrations of morphine and (ii) supersensitivity to the excitatory APD-prolonging effects of nanomolar NLX as well as of ultra-low (femtomolar-picomolar) concentrations of morphine and other opioid agonists. These in vitro studies suggested that ultra-low doses of NLX or NTX that selectively block the excitatory effects of morphine may not only enhance the analgesic potency of morphine and other bimodally acting opioid agonists but also markedly attenuate their dependence liability. Subsequent correlative studies have now demonstrated that cotreatment of mice with morphine plus ultra-low-dose NTX does, in fact, enhance the antinociceptive potency of morphine in tail-flick assays and attenuate development of withdrawal symptoms in chronic, as well as acute, physical dependence assays.

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Electrical investigation of p-type 4H-SiC heavily doped by ion implantation

In the last decades, an increasing interest in the research field of wide bandgap semiconductors was observed, mostly due to the progressive approaching of silicon-based devices to their theoretical limits. 4H-SiC is an example among these, and is a mature compound for applications. The main advantages offered 4H-SiC in comparison with silicon are an higher breakdown field, an higher thermal conductivity, a higher operating temperature, very high hardness and melting point, biocompatibility, but also low switching losses in high frequencies applications and lower on-resistances in unipolar devices. Then, 4H-SiC power devices offer great performance improvement; moreover, they can work in hostile environments where silicon power devices cannot function. Ion implantation technology is a key process in the fabrication of almost all kinds of SiC devices, owing to the advantage of a spatially selective doping. This work is dedicated to the electrical investigation of several differently-processed 4H-SiC ion- implanted samples, mainly through Hall effect and space charge spectroscopy experiments. It was also developed the automatic control (Labview) of several experiments. In the work, the effectiveness of high temperature post-implant thermal treatments (up to 2000°C) were studied and compared considering: (i) different methods, (ii) different temperatures and (iii) different duration of the annealing process. Preliminary p + /n and Schottky junctions were also investigated as simple test devices. 1) Heavy doping by ion implantation of single off-axis 4H-SiC layers The electrical investigation is one of the most important characterization of ion-implanted samples, which must be submitted to mandatory post-implant thermal treatment in order to both (i) recover the lattice after ion bombardment, and (ii) address the implanted impurities into lattice sites so that they can effectively act as dopants. Electrical investigation can give fundamental information on the efficiency of the electrical impurity activation. To understand the results of the research it should be noted that: (a) To realize good ohmic contacts it is necessary to obtain spatially defined highly doped regions, which must have conductivity as low as possible. (b) It has been shown that the electrical activation efficiency and the electrical conductivity increase with the annealing temperature increasing. (c) To maximize the layer conductivity, temperatures around 1700°C are generally used and implantation density high till to 10 21 cm -3 . In this work, an original approach, different from (c), is explored by the using very high annealing temperature, around 2000°C, on samples of Al + -implant concentration of the order of 10 20 cm -3 . Several Al + -implanted 4H-SiC samples, resulting of p-type conductivity, were investigated, with a nominal density varying in the range of about 1-5∙10 20 cm -3 and subjected to two different high temperature thermal treatments. One annealing method uses a radiofrequency heated furnace till to 1950°C (Conventional Annealing, CA), the other exploits a microwave field, providing a fast heating rate up to 2000°C (Micro-Wave Annealing, MWA). In this contest, mainly ion implanted p-type samples were investigated, both off-axis and on-axis <0001> semi-insulating 4H-SiC. Concerning p-type off-axis samples, a high electrical activation of implanted Al (50-70%) and a compensation ratio below 10% were estimated. In the work, the main sample processing parameters have been varied, as the implant temperature, CA annealing duration, and heating/cooling rates, and the best values assessed. MWA method leads to higher hole density and lower mobility than CA in equivalent ion implanted layers, resulting in lower resistivity, probably related to the 50°C higher annealing temperature. An optimal duration of the CA treatment was estimated in about 12-13 minutes. A RT resistivity on the lowest reported in literature for this kind of samples, has been obtained. 2) Low resistivity data: variable range hopping Notwithstanding the heavy p-type doping levels, the carrier density remained less than the critical one required for a semiconductor to metal transition. However, the high carrier densities obtained was enough to trigger a low temperature impurity band (IB) conduction. In the heaviest doped samples, such a conduction mechanism persists till to RT, without significantly prejudice the mobility values. This feature can have an interesting technological fall, because it guarantee a nearly temperature- independent carrier density, it being not affected by freeze-out effects. The usual transport mechanism occurring in the IB conduction is the nearest neighbor hopping: such a regime is effectively consistent with the resistivity temperature behavior of the lowest doped samples. In the heavier doped samples, however, a trend of the resistivity data compatible with a variable range hopping (VRH) conduction has been pointed out, here highlighted for the first time in p-type 4H-SiC. Even more: in the heaviest doped samples, and in particular, in those annealed by MWA, the temperature dependence of the resistivity data is consistent with a reduced dimensionality (2D) of the VRH conduction. In these samples, TEM investigation pointed out faulted dislocation loops in the basal plane, whose average spacing along the c-axis is comparable with the optimal length of the hops in the VRH transport. This result suggested the assignment of such a peculiar behavior to a kind of spatial confinement into a plane of the carrier hops. 3) Test device the p + -n junction In the last part of the work, the electrical properties of 4H-SiC diodes were also studied. In this case, a heavy Al + ion implantation was realized on n-type epilayers, according to the technological process applied for final devices. Good rectification properties was shown from these preliminary devices in their current-voltage characteristics. Admittance spectroscopy and deep level transient spectroscopy measurements showed the presence of electrically active defects other than the dopants ones, induced in the active region of the diodes by ion implantation. A critical comparison with the literature of these defects was performed. Preliminary to such an investigation, it was assessed the experimental set up for the admittance spectroscopy and current-voltage investigation and the automatic control of these measurements.

Eficiência de fontes, dinâmica do magnésio no solo sob a cultura do café no Brasil e comparação da análise de K, Ca e Mg em solos do Arkansas-EUA seco em estufa e úmido de campo

A cultura do café no Brasil tem apresentado frequente deficiência de magnésio (Mg) limitando sua produtividade, portanto faz-se necessário o estudo de fontes que contenham Mg para essa cultura. Por outro lado, o estudo das metodologias de análise de K, Ca e Mg no solo é um outro ponto que precisa ser estudado para melhor manejo da fertilidade do solo e recomendação de adubações. Objetivou-se com o primeiro experimento avaliar a eficiência de fontes de magnésio para a cultura do café e a dinâmica deste nutriente no perfil do solo. E com o experimento desenvolvido em Arkansas-EUA, avaliar as correlações entre as concentrações de nutrientes do solo seco em estufa e úmido de campo extraídos com Mehlich-3 e 1 mol L-1 NH4OAc. Observou-se que o óxido e oxissulfato de Mg elevaram os valores de pH e CTC e diminuíram a concentração de H + Al do solo. As fontes diminuíram a disponibilidade de K e Ca, e aumentaram o Mg no solo. Na planta, óxido e sulfato de Mg proporcionaram maior concentração de Mg foliar. Apenas no segundo ano de avaliação houve aumento de produtividade do café. Os fertilizantes óxido e oxissulfato de Mg obtiveram o maior índice de eficiência agronômica em relação ao carbonato de Mg. No segundo experimento, K, Ca e Mg extraíveis com Mehlich-3 e NH4OAc foram altamente correlacionados (r2> 0,95) tanto para solo úmido de campo quanto para o seco em estufa. A relação entre as concentrações de K no solo seco em estufa e úmido de campo para Mehlich-3 e NH4OAc foram muito semelhantes e altamente correlacionados (r2 = 0,92). A secagem do solo em estufa teve efeito mínimo sobre as concentrações de Ca e reduziu a concentração de Mg tanto para Mehlich-3 quanto para NH4OAc. Entre os nutrientes estudados, a concentração de K foi a mais afetada pela secagem em estufa, necessitando de pesquisas de campo para correlacionar e calibrar novas recomendações agronômicas.

Simultaneous optical and X-ray observations of flares and rotational modulation on the RS CVn binary HR 1099 (V711 Tau) from the MUSICOS 1998 campaign

We present simultaneous and continuous observations of the Hα, Hβ, He I D_3, Na I D_1,D_2 doublet and the Ca II H&K lines for the RS CVn system HR 1099. The spectroscopic observations were obtained during the MUSICOS 1998 campaign involving several observatories and instruments, both echelle and long-slit spectrographs. During this campaign, HR 1099 was observed almost continuously for more than 8 orbits of 2^d.8. Two large optical flares were observed, both showing an increase in the emission of Hα, Ca II H K, Hβ and He I D_3 and a strong filling-in of the Na I D_1, D_2 doublet. Contemporary photometric observations were carried out with the robotic telescopes APT-80 of Catania and Phoenix-25 of Fairborn Observatories. Maps of the distribution of the spotted regions on the photosphere of the binary components were derived using the Maximum Entropy and Tikhonov photometric regularization criteria. Rotational modulation was observed in Hα and He I D_3 in anti-correlation with the photometric light curves. Both flares occurred at the same binary phase (0.85), suggesting that these events took place in the same active region. Simultaneous X-ray observations, performed by ASM on board RXTE, show several flare-like events, some of which correlate well with the observed optical flares. Rotational modulation in the X-ray light curve has been detected with minimum flux when the less active G5 V star was in front. A possible periodicity in the X-ray flare-like events was also found.