We employed transgenic mice with fluorescently labeled NG2 cells toaddress this issue. To identify Ca2+-signaling pathways wecombined patch-clamp recordings, Ca2+-imaging, mRNA-transcriptanalysis and focal pressure-application of various substances to identifiedNG2-cells in acute hippocampal slices. We show that activation of voltage-gatedCa2+-channels, Ca2+-permeableAMPA-receptors, and group I metabotropic glutamate-receptors provoke[Ca2+]i-elevations in NG2 cells. TheCa2+-influx is amplified by Ca2+-inducedCa2+-release. Minimal electrical stimulation of presynapticneurons caused postsynaptic currents but no somatic[Ca2+]i elevations, suggesting that[Ca2+]i elevations in NG2 cells mightbe restricted to their processes. Local Ca2+-signaling mightprovoke transmitter release or changes in cell motility. To identify structuralprerequisites for such a scenario, we used electron microscopy, immunostaining,mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells formsymmetric and asymmetric synapses with presynaptic neurons and showimmunoreactivity for vesicular glutamate transporter 1. The processes areactin-based, contain ezrin but not glial filaments, microtubules or endoplasmicreticulum. Furthermore, we demonstrate that NG2 cell processes in situ arehighly motile. Our findings demonstrate that gray matter NG2 cells are endowedwith the cellular machinery for two-way communication with neighboringcells.NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic inputfrom neurons. The function of this innervation is unknown yet. Postsynapticchanges in intracellular Ca In addition to astrocytes, oligodendrocytes, and microglia, NG2 cells are nowrecognized as a fourth glial cell type in the CNS 2+-elevations in post-synaptic NG2 cells, which might evokecellular motility or release of neuroactive substances. This ignorance is quiteastonishing in view of the increasing knowledge of glia-mediated modulation of CNSsignaling, such as astrocyte-neuron interactions which gave rise to the tripartitesynapse concept 2+-permeable AMPA receptors 2+-channels (Cavs) vs are activated by the depolarizing action of GABAvs in NG2 gliais still disputed. Instead, a role for the Na+-Ca2+exchanger (NCX) in NG2 cell Ca2+-signaling has recently beenproposed NG2 cells are unique among glial cells in receiving synaptic input  generate transient elevations of theintracellular Ca2+-concentration([Ca2+]i) upon different types ofstimulation and (ii) display in situ highly motile actin-basedprocesses.There are different terms in the literature describing NG2-like cells in acutepreparations of wild type or different transgenic mouse lines: complex glial cellse.g. ;GluR ce2+-imaging  were EYFP positive, had an input resistance of 193±157MΩ, a resting membrane potential of −77±6 mV, and a membranecapacity of 33±8 pF (K+-based pipette solution). Allcells tested (n = 23) received glutamatergic and/orGABAergic synaptic input (not shown). EYFP positive cells from homozygous(n = 351) and heterozygous (n = 340)mice did not differ with respect to the above membrane parameters, expression ofCav channel transcripts, and Ca2+-responsivenessupon somatic depolarization or high frequency stimulation of pre-synaptic fibers(see below for details). Therefore, data were pooled.Cell identification in the hippocampus was based on EYFP or EGFP fluorescence,morphology, and physiological criteria as reported previously 1, E1). Inseveral axon terminals, docked vesicles were observed at the pre-synapticmembrane . In some cases, the DAB reactionproduct was faint enough to reveal distinct post-synaptic detail, which wasindistinguishable from neuron-neuron synapses. Thus, several neuron-NG2 cellsynapses could be unequivocally classified as either asymmetric (7/17) orsymmetric (1/17)  are shown in membrane 1,2, E1./17) see . All syn/17) see , but in /17) see . Thus, i2+-signaling in post-synapticNG2 cells. Therefore, we tested for potential pathways provoking[Ca2+]i elevation in NG2 cells, whichmight be activated by the synaptic input.The physiological properties of these neuron-NG2 cell-synapses are characterizedin some detail vvcurrents were isolated using Na+- and K+-freebath and pipette solutions. In addition, solutions were supplemented withNav and Kv channel blockers, and[Ca2+] in the bath was increased to 5 mM  (1). Plotting the I/V relationship of the evoked currentsrevealed a threshold potential of −60 mV, while peak inward currentsoccurred at about −20 mV (2). The L-type channel blocker Verapamil (100µM) reduced the maximum inward currents from 167±35 pA to85±33 pA  and significantlyshifted the half maximum voltage of the steady state inactivation curve . Coapplication of theT-type channel blocker Mibefradil (50 µM) further diminishedCav currents in 4/5 cells tested (to 25±10 pA). Theseproperties resemble Cav currents in complex glial cells of thehippocampal CA1 region Previous work has demonstrated that complex glial cells in wild type mice expressdifferent types of Ca5 mM see. Toremon = 14) 1. Plottt −20 mV 2. The LA n = 9, 2 and sivs expressed by NG2/EYFP positivecells, transcript analysis was performed employing single cell RT-PCR (v 1.2 and Cav 1.3 (1) and the T-type channelsCav 3.1 and Cav 3.2. Transcripts for P/Q and N-typechannels, Cav 2.1 and Cav 2.2, were less abundant, whilemRNAs for Cav 1.4, Cav 2.3 and Cav 3.3 werenever detected (2). Interestingly, the majority of NG2 cells tested(n = 39/46) expressed mRNA for the glial marker S100βThis is in line with our previous data showing that some of the NG2/EYFPpositive cells express S100β while the astrocytic marker GFAP wasconsistently lacking .To identify the subtype(s) of Cal RT-PCR .We foun Cav 1.3 1 and thdetected 2. Intervs in NG2 cells of thehippocampus, Ca2+-imaging was combined with patch-clamprecording in the whole-cell mode. Train stimulation via the patch-pipette (15consecutive depolarizing voltage steps (100 ms) from −100 mV to +20mV, see lower traces in 1) produced reversible elevations of[Ca2+]i in NG2/EYFP cells (1). It isimportant to note that in the same cell, several[Ca2+]i elevations could be elicited upto 30 min after establishing the whole-cell configuration (2). Next, we tested thesensitivity of the [Ca2+]i elevations toNi2+. At high concentrations Ni2+ is knownto non-specifically block Cavs 2+ abolished the[Ca2+]i elevations in the NG2/EYFPcells tested (n = 4)  .2+ might also inhibit the NCX2+]ielevations by Ni2+ was due to its action on NCX rather thanCavs, we tested the sensitivity of evoked[Ca2+]i elevations to the NCX inhibitorSN-6. SN-6 has no effect on Cavs while blocking NCX operating in theCa2+-influx mode v currents(at −10 mV) were not affected by SN-6 . The[Ca2+]i elevation immediately ceasedafter the last pulse (1). In contrast,[Ca2+]i elevations by a single pulseconsiderably outlasted the pulse duration. Maximal[Ca2+]i was observed about 1.2 s afterstimulus offset. During this timeΔ[Ca2+]i almost doubled .We further analyzed the kinetics and amplitudes of depolarization-inducedi elevation were almost the sameusing either imaging technique . Obviously, there was aceiling effect because the [Ca2+]ielevations during train stimulation were much smaller than the calculatedsuperposition of the responses to 15 single pulses  sensitive manner 2+]ielevations in NG2/EYFP cells  isoperative in NG2 cells we performed recordings in nominalCa2+-free bath solution supplemented with 2 mM EDTA. Under theseconditions no [Ca2+]i elevation could beelicited by train stimulation. The same individual cells showed strong increasesin [Ca2+]i after switching to artificialcerebrospinal fluid (aCSF) bath solution containing 2 mM Ca2+ , a blocker of sarco/endoplasmic reticulumCa2+-ATPase 2+]i elevations declined to16% of the control value (n = 5)  . Hence,  (n = 5) . This su−]i in NG2cells, activation of GABAA receptors has a depolarizing effect A receptor agonists induce elevationsin [Ca2+]i in NG2/EYFP cells. TTX (1µM) was added to the bath solution to reduce indirect effects. In thecurrent-clamp mode, the AMPA/kainate receptor agonist kainate  as well as the GABAA receptor agonistmuscimol  induced[Ca2+]i elevations (2+]i (1), due to activation ofCa2+-permeable AMPA receptors 2+]i (n = 4)(2).These data demonstrate that AMPA/kainate receptor activation may produce direct(Ca2+-influx through the receptor pore or possibly throughmetabotropic effects vs followed by Ca2+-influx)[Ca2+]i elevations. In contrast,GABAA receptor-induced Ca2+-influx in NG2 cellsis indirect, i.e. due to membrane depolarization and Cavactivation.Due to a relatively high [Clevations . In the ns 2.These2+]i weremonitored by simultaneous patch-clamp recording in the whole cell mode and linescan imaging. All cells tested responded to 3,5-DHPG with[Ca2+]i elevation . This was never accompanied by currentresponses (1). The delay between substance arrival and the onset of[Ca2+]i rises  varied among cells , but not between multiple 3,5-DHPG applications tothe same individual cell.Next, we tested whether NG2 cells express metabotropic glutamate receptors(mGluRs). The group I mGluR-specific agonist 3,5-DHPG was focally applied, whilemembrane currents and [Caesponses 1. The d2+]i elevations. Although indicating the involvement of mGluRs, theseresponses might have been produced indirectly, i.e. via mGluR activation ofneighboring cells that innervate the NG2 cell. Another constraint of theseexperiments was the significant run down of the[Ca2+]i elevations upon repetitive3,5-DHPG applications , probably due to wash-out of cytosolicconstituents during whole cell recording. To circumvent these limitations weadded TTX (1 µM) to block action potentials and inhibited P2Y receptors, mACh receptors (with 5 µMipratropium), 5-HT2 receptors (with 10 µM methysergide), α1 receptors(with 10 µM prazosin), and GABAB receptors (with 2 µMCGP55845). In addition, local loading of groups of NG2/EYFP cells with Fluo-4 AMwas employed using focal pressure application . Underthese conditions, almost all NG2 cells tested (96%) showed robust[Ca2+]i elevations upon application of3,5-DHPG . Furtheranalysis using mGluR group I subtype-specific antagonists , while the mGluR5antagonist MPEP (20 µM) abolished[Ca2+]i elevations in 74% of thecells . Inthe remaining cells, 3-MATIDA (n = 8) and MPEP(n = 6) exerted partial inhibition of 3,5-DHPG-inducedresponses (to 47±17%) that did not differ significantly betweenthe antagonists. Co-application of both antagonists abolished[Ca2+]i elevations in 88% of theNG2 cells tested (n = 15/17) (3). The[Ca2+]i transients recovered after washout of the antagonists to 78±23% (n = 47) ofthe initial value. We noted that all cells were sensitive to at least one of thetwo antagonists.Pre-application of the unspecific group I mGluR antagonist LY341495 sn = 2, 2. Altholication 1, B2. U= 15/17) 3. The[2+]i elevations in NG2 cells.Minimal stimulation induced post-synaptic currents in NG2/EYFP cells matchingthose observed in weakly fluorescent hGFAP/EGFP cells (previously termed GluRcells) 2+]i . Tosimulate more physiological conditions, single pulses (200 µs) wereapplied. With this protocol, a failure rate of about 60% was observed.Excluding failures, the post-synaptic depolarization now amounted to1.5±0.6 mV .These depolarizations were never accompanied by somatic[Ca2+]i elevations(n = 12)  . Obvious2+]ielevations prompted us to search for potential downstream signaling mechanismsin NG2 cells. Astrocytes express vesicular glutamate transporters (vGLUTs) intheir distal processes, and were reported to communicate with neurons byCa2+-dependent release of vesicular glutamate The observation of stimulus-induced [Ca2+]i elevation and migration of NG2cells in vitroin situ, weperformed time-lapse recordings in acute hippocampal slices. We detected processmotility in 5 out of 11 dye-labeled NG2/EYFP cells  miceand selected according to their characteristic morphology and specificimmunolabeling  2+]i via several independentpathways: G-protein coupled receptors, as well as ligand- and voltage-gatedion-channels. While the presence of mGluRs in NG2 cells represents a newfinding, expression of Cavs is under discussion. Recently, it wasreported that NG2 cells in the hippocampus lack Cavs vs which were sensitive toCd2+ or dihydropyridines and omega-conotoxin GIVA,respectively vs inidentified NG2/EYFP cells. This discrepancy with the former data may be due todifferent recording conditions. Ca2+-currents in NG2 cells aresmall in amplitude, compared with the dominating K+ currents.Its reliable separation requires use of Na+- andK+-free solutions, elevated [Ca2+]in the bath solution and application of conditioning pre-pulses.Our data demonstrate the capability of gray matter NG2 cells to increasei elevations in NG2 cells. Thiswas due to influx of Ca2+ through Cavs, but not tothe activation of NCXs, as recently suggested vs with almost the sameaffinity 2+ does not only block Cavs but alsoNXCs 2+-influx mode of NCXs, preferentially of NCX1, while notinterfering with Cavs at the concentration used here 2+-currents evations  we belie2+]i elevation through Cavactivation was almost doubled due to CICR. Notably, this led also to asignificant prolongation of the [Ca2+]ielevations. Thus, CICR represents a powerful mechanism to amplify small inwardcurrents through Cavs in NG2 cells. The observed saturation effect filaments or microtubules were not observedby immunolabeling and electron microscopy. This appears astonishing in respectof their length (30–50 µm) and small diameter (0.2–1 µm)in between the varicose expansions. Of the many actin-binding proteins ezrin waschosen as a further marker, because its (de)phosphorylation-based mode ofmembrane-to-cytoskeleton linking enables rapid shape changes Our live microscopic data demonstrate, for the first time, motility of NG2 cellprocesses 2+]i elevation is essential formigration of subventricular zone NG2 cells to and within white matter invitrovs might be important in this context as they have beenreported to govern migration in newborn neurons, e.g. in the postnatal olfactorybulb Recent findings suggest a role of neuron-NG2 cell synapses in migration. Thus, inthe corpus callosum adult-born migrating NG2 cells receive glutamatergicsynaptic input from demyelinated axons 2+]ielevations through the Ca2+-signaling pathways reported here.However, because the processes are devoid of endoplasmic reticulum, these[Ca2+]i elevations are unlikely to beamplified by CICR and might occur locally confined. Local[Ca2+]i elevations might play a role inregulation of process motility. In addition, restrictedCa2+-signaling might be interesting in the light of thedemonstrated vGLUT expression. In neurons, vGLUT expression is sufficient fordefining a glutamatergic phenotype 2+-dependent manner remains to be addressed infuture studies.Synaptic activation may cause small i and achieve a high timeresolution of Ca2+-transients two different methods wereapplied.NG2/EYFP cells in the stratum radiatum of the CA1 area were used forCa2+]i were monitored by aCCD camera  mounted on awide-field epifluorescence system . It wasattached to an upright microscope equipped with a 60x LUMPlan FI/IR objective . Fluorescence excitation was achieved by a monochromator. Individualcells in acute hippocampal slices were loaded via the patch-pipette with Fura-2. Dye filling lasted ≥5 min beforeCa2+-imaging was started. If not stated otherwise, Fura-2was excited at 380 or 340 nm for 40 ms and emission was detected at anacquisition rate of 25 Hz during, and 3 Hz after depolarization. Single frameswere recorded at the isosbestic point (362 nm) before and after each sequence.This allowed offline calculation of pseudo-ratiometric images to correct forbleaching. The latter was assumed to be proportional to exposure time. A linearfunction was calculated from the first and the last 362 nm frame of each of the380 or 340 nm sequences. This function was used to determine the 362 nm valuesfor each recorded frame. Pseudo-ratios F380 orF340/F362 were calculated from the measuredF380 or F340 and the extrapolated F362values for each time point. F380/F362 pseudo-ratios wereinversely plotted so that [Ca2+]ielevations are always indicated by upward deflections.(i) Changes in [Ca2+]i was estimated throughcalibration according to Grynkiewicz et al. Absolute i,measured as change in fluorescence intensity (ΔF), were offline related tothe baseline fluorescence (F0) according to ΔF/F0 = (F - F0)/F0. Time-correlatedsignals from individual cells were averaged to improve signal-to-noise ratio.For local loading of groups of EYFP positive cells, Fluo-4 AM  with 0.01% Pluronic F127 was focally pressure-applied for 5min employing an Octaflow System (ALA Scientific Instruments). x-y-t scans of2.2 µm thick single optical planes were recorded. ΔF/F0 wasdetermined in separate regions of interest (ROIs) placed in each NG2 cell somain the field of view. Data analysis was performed with LAS Live Data Mode(Leica) and IgorPro 6 software. 3-MATIDA, (S)-3,5-DHPG, CGP 55845, GABA,ipratropium, kainic acid, methysergide, MPEP, muscimol, PPADS, prazosin, SN-6,suramin, and thapsigargin were from Tocris (ii) Alternatively, an LSM (Leica) was used for CaStimulation was performed with monopolar glass pipettes filled with aCSF. Pipetteresistance ranged between 0.5 and 2 MΩ Biphasic constant voltage-pulses of100–200 µs were applied with a stimulus generator . High-frequency stimulation wasaccomplished using Mc Stimulus 2 software (Multi-Channel-Systems). Timecorrelation was achieved by synchronizing TTL pulses generated by the recordingsoftware .in situ, thecytoplasm of individual cells was harvested under microscopic control asreported previously v 1, Cav 2and Cav 3 families or vesicular glutamate transporters (vGLUT) 1/2and vGLUT3, respectively (2 (2.5 mM), and primers (200nM each) to the reverse transcription product . Taqpolymerase  was added after denaturation. 45 cycles wereperformed . An aliquot (2 µl) of the PCR product wasused as a template for the second PCR  using nested, subunit-specificprimers . For optimization, a two-round RT-PCR was performed with 2 ng of total RNAand primers as described above. Subsequent gel analysis did not detectunspecific products. The primers for different targets were located on differentexons to prevent amplification of genomic DNA. Omission of the RT-enzyme andsubstitution of template by bath solution served as negative controls forreverse transcription and PCR amplification and confirmed the specificity of thereaction.2O2, forultrastructural staining. Sections were osmicated (1% OsO4), blockstained (1% uranyl acetate in 70% ethanol), dehydrated and flatembedded in Araldite. Ultrathin sections were contrasted with lead citrate anduranyl acetate. To analyze overall synaptic contacts on NG2 cells at theultrastructural level, these flat embedded cells were completely sectioned.Inspecting all ultrathin sections from a given cell, the complete process treewas scanned for synapses on DAB-containing profiles. Most synapses found in onesection could also be documented in subsequent sections. To estimate the totalnumber of synapses, the observed number of synapses was documented  hGFAP-EGFPmice. Weakly fluorescent cells with a typical electrophysiologicalcurrent-pattern . Subsequently, sections weredisaggregated using pipettes, centrifuged, and resuspended in inhibitor solution. Finally, the cells were centrifuged onto silane-coatedslides and immediately fixed with 4% PFA.The isolation method applied adapts previous cell-isolation protocols Dissociated cells on slides were quadruple-stained; incubation was with a mixtureof the three primary or secondary antibodies according to standard procedures.The primary antibodies were chicken anti-GFAP , sheep anti-GFP , and a label for the protein of interest, viz. mouse anti-ezrin, mouse anti-α-tubulin, mouse anti-β-actin , or rabbitanti-PDI . For cellidentification and nucleus localization, AMCA-coupled donkey anti-chicken(1∶100) and dylight488-coupled donkey anti-sheep (1∶100) werecombined with bisbenzimidine . For visualization of the antigenof interest, cells were incubated with CY3 coupled to donkey anti-rabbit(1∶250) or anti-mouse (1∶250). NG2 cell identification was based onmorphology , presence of staining with anti-GFP but absence of staining withanti-GFAP in situ,vGLUT1 or vGLUT2 immunofluorescence was combined with fluorescence detection ofbiotycin-filled NG2 cells. Cells were identified and filled as above, and fixedin 4% PFA . After freeze-thawing, the sections were incubatedwith streptavidin-CY3 . Subsequent immunostaining wascarried out by incubating sequentially with normal goat serum , rabbit anti-vGLUT2 , and goat anti-rabbit-Alexa 647 . Forvisualization of vGLUT1, only rabbit anti-vGLUT1 directly coupled to Oyster-645 was applied overnight.To detect putative glutamate vesicles in NG2 cells Detection of vGLUT-IR in NG2 cells is challenging because it is abundant anddense in brain, and NG2 cell processes are frequently thinner than 0.5 µm,as observed in the electron microscope cf. . We carrFigure S1Microtubules are well-preserved in the processes of freshlydissociated, identified astrocytes. Labeling for both, cellnuclei (bisbenzimidine) and glial filaments  is revealed inthe blue channel. An astrocyte (center) and two unidentified cells (right)are displayed. Microtubules  are obvious in theastrocyte processes demonstrating that the dissociation method does notinterfere with microtubule integrity even in the processes.(TIF)Click here for additional data file.Figure S2v channelfamily and S100β.Exemplary agarose gels of mRNA-transcripts for Ca(TIF)Click here for additional data file.Table S1Primers used for single-cell RT-PCR.(DOC)Click here for additional data file.Video S1Demonstration of full inclusion of vGLUT1 positive objects in NG2 cellprocesses (3D reconstruction). The cell is the one shown in (AVI)Click here for additional data file.Video S2Elongation of an NG2 cell process. (cf. ess. cf. . Two-pho(AVI)Click here for additional data file.Video S3Retraction of an NG2 cell process and movement of intracellularvaricosities. (cf. ies. cf. . Similar(AVI)Click here for additional data file.


The mechanisms of Ca2+-ATPases dysfunction are discussed, focusing on cystein oxidation and tyrosine nitration. Flavonoids act not only as antioxidants but are also able to bind directly to Ca2+-ATPases, thus changing their conformation, which results in modulation of enzyme activity.Flavonoids, natural phenolic compounds, are known as agents with strong antioxidant properties. In many diseases associated with oxidative/nitrosative stress and aging they provide multiple biological health benefits. Ca2+-ATPases is summarized with respect to their posttranslational and conformational changes in diseases related to oxidative/nitrosative stress and aging. Ca2+-ATPases are discussed as a therapeutic tool and the possible role of flavonoids in this process is suggested.Dysfunction of Ca In addition, modulation of Ca2+-ATPases by antioxidants may be a therapeutic tool for improvement of complications associated with human diseases and their experimental animal models. Flavonoids, in addition to their antioxidant properties exert beneficial effects based on their ability to bind to Ca2+-ATPases and thus to change their activity.Free oxygen and nitrogen reactive species  are participating in aging and age related diseases. The search for mechanisms of oxidative/nitrosative injury involved in diseases and aging is important for finding new therapeutic approaches. Impairment of calcium homeostasis is a consequence of oxidative/nitrosative stress. In recent years, many results were published showing that dysfunction of Ca2+-ATPases, their impairment and impact as a possible therapeutic tool in diseases related to oxidative/nitrosative stress has not yet been summarized. This review deals with the modulation of Ca2+-ATPases by oxidative/nitrosative stress in vitro and in diseases and with their potential therapeutic effect.The importance of Caet al., et al., i.e. the modulation of calcium homeostasis and cell signaling via Ca2+-ATPase.Flavonoids are phenolic compounds known as characteristic red, blue and purple anthocyanin pigments of plant tissues . The polymers of catechins are excellent in vitro antioxidants due to the high number of hydroxyl groups. The glycosylation of flavonoids reduces their in vitro antioxidant activity, compared with corresponding aglycones  an ortho-dihydroxy (catechol)structure in the B ring for electron delocalization, ii) 2,3-double bond in conjugation with a 4-oxo function in the C ring providing electron delocalization from the B ring, iii) hydroxyl groups at positions 3 and 5 providing hydrogen bonding to the oxo group Croft  Figure .et al., et al., et al., The ability to interact with various antioxidant enzymes belongs also to important mechanisms of flavonoids in preventing injury caused by free radicals. Protective effects of quercetin and catechin against hydrogen peroxide toxicity in cultured rat hepatocytes BL-9 were related to the activation of glutathione peroxidase (GPx) , yet they did not inhibit its activity. Several flavonoids, including quercetin, induce a reduction of ischemia-reperfusion injury by interfering with inducible NOS activity . GSH, when oxidized, is rapidly reduced and/or resynthetized to protect proteins from oxidation. With RNS GSH forms, S-nitroso glutathione (GSNO), sulfenic acid (GSOH), and GSH thiyl radicals (GS.), which can react with nearby protein thiols. Protein thiolate anions (R-S–) are predisposed to react with these species. Chemical adducts of protein thiols generated in the presence of RNS include Pr-SNO (generated by S-nitrosylation), Pr-SSG (generated by glutathiolation) and oxidized thiols (Pr-SOH), (Pr-SO2H), (PrSO3H), as well as intra- and inter-protein dithiols. All modifications, except those of sulfinic acid (Pr-SO2H) and sulfonic acid (PrSO3H), are reversible under physiologic conditions, enabling them to participate in cell signaling.The mechanisms of intracellular calcium regulation by SERCA, involved in vascular relaxation, in cardiac and muscle relaxation, as well as in growth and differentiation, may include peroxinitrite (generated from nitric oxide and superoxide), which causes glutathione (GSH) to bind to SERCA cysteine thiols (Cohen 2 adduct of peroxynitrite (ONOO-) or of NO2  rapidly homolyzes to •CO–3  and.NO2 (nitrogen dioxide) to undergo aradical mechanism of tyrosine nitration. This involves hydrogen atom abstraction from the 3-position of tyrosine to form the tyrosyl radical that rapidly combines with.NO2 forming 3-nitrotyrosine. Protein nitration may also be formed by generation of •NO2 by heme peroxidases, such asmyeloperoxidase and eosinophil peroxidase, in the presence of hydrogen peroxide.Another peroxynitrite product is nitrotyrosine. In SERCA2 in the aging heart and skeletal muscle nitration of vicinal tyrosines, Tyr 294 and Tyr295 was demonstrated on the luminal side of the membrane-spanning helix M4, correlating with partial inhibition of Ca-ATPase activity. This is suggesting a possible regulatory function in down-regulating mitochondrial energy production and the associated decrease in generation of reactive oxygen/nitrogen species Bigelow, 2007). Nitrotyrosine is generated from nitric-oxide-derived oxidants, mainly of the CO. Nitrotyin vivo, whereas higher concentratioins of peroxynitrite result in irreversible modification of Cys674 to cystein sulfonic acid (Cys-SO3H) and accompanying nitrotyrosine modification , and different SERCA isoforms are expressed in tissue-specific and developmentally regulated patterns  headpiece. Ca-binding sites are located within the membrane-bound region, the ATP binding (nucleotide binding) site is situated in the cytosolic region of SERCA .SERCA pumps play a central role in maintaining low levels of free cytosolic calcium within cells. This Ca2+-ATPase activity at least partially by structural changes, was prevented by standardized flavonoid extract from leaves of Ginkgo biloba (EGb761)  prevented sulfhydryl (SH) group oxidation and protein carbonyl (PCO) formation. Pyc in addition prevented thiobarbituric acid reactive species (TBARS) formation in SR oxidized by the Fenton system. The lower antioxidant effect of EGb761 compared with Pyc may be associated with the fact that OH-groups of compounds included in this extract and effective in scavenging free radicals are bound to glucose Drieu, . Mixturea et al. . Flavonoa et al. . EGb761 2+-ATPase activity by antioxidants depends on the mode of oxidative injury. We further suggest that conformational alterations in cytosolic nucleotide (ATP)-binding or transmembrane Ca2+-binding regions of Ca2+-ATPase, which are now under investigation in our laboratory, may be also critical for protective effects of antioxidants.We conclude that the ability to protect Caet al., et al., et al., et al., –-mediated SERCA injury at low concentrations (5–50 µmol/l) and inhibitory at higher concentrations (100–250µmol/l), , epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC). The chemical structure of catechins is shown in et al., +,K+-ATPase in human erythrocyte ghosts . Daily intake of EGCG by drinking green tea may provide physiological benefits to humans via inhibition of Na+,K+-ATPase. EGCG and ECG exert their inhibitory effect on the membrane-bound Na+,K+-ATPase allosterically through interaction with plasma membrane phospholipid. The two galloyl-type catechins (EGCg and ECg) showed a stronger inhibitory effect on Na+,K+-ATPase than the other two catechins lacking galloyl groups (EGC and EC). EGCg and ECG have a greater affinity for membrane phospholipid than EGC and EC  can inhibit the activity of Ca2+-ATPases isoforms SERCA1A and SERCA2B in the micromolar concentration range. Of the 25 flavonoids tested, 3,6-dihydroxyflavone  and 3,30,40,5,7-pentahydroxyflavone (quercetin)  were the most potent inhibitors. The polyhydroxylation of flavones is important for inhibition, with hydroxylation at position 3 (for SERCA1A) and position 6 (for SERCA2B) being particularly relevant . Pyc is able to inhibit SERCA  studied the effects of combining EGCG and anticancer drugs, focusing on inhibition of cell growth and induction of apoptosis. Synergistic enhancement of apoptosis, gene expression, and anticancer effects were found on using various combinations of EGCG and anticancer drugs, including the combination of (-)-epicatechin (EC) and curcumin  may help protect the aging brain and reduce the incidence of dementia like Alzheimer′s disease (AD) and Parkinson′s disease (PD). Neuroprotective/neuroregenerative effects of green tea catechins were found to act not only as antioxidant metal chelators but also as modulators of intracellular neuronal signaling and metabolism, of cell survival/death genes, and mitochondrial function. Thus, these dietary compounds are receiving significant attention as therapeutic multifunctional cytoprotective agents that simultaneously manipulate various brain targets .Many antioxidants have shown inhibitory effects on cardiac hypertrophy et al.  homeostasis is another important cause for doxorubicin-mediated cardiotoxicity. Precise Ca2+ handling in cardiomyocytes is crucial for excitation–contraction coupling, the process that enables the heart to function effectively as a pump. Doxorubicin was reported to regulate the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, SERCA2 gene transcription and ryanodine receptors, thus modulating heart contractile function.  function and Ca2+ homeostasis may be involved in the etiology of ischemia/reperfusion injury. Ischemia–induced structural changes in SR Ca2+-ATPase are associated with reduced enzyme activity in rat muscle. Experiments with prolonged ischemia in rat skeletal muscle revealed that structural modifications to SR Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia . They found that reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia was related to structural modification in the region of the nucleotide binding domain.Conformational alterations of Ca2+ into SR or ER  to SERCA cysteine thiols. The reaction of NO with superoxide anion is required, as indicated by the fact that mouse arteries in which superoxide dismutase is overexpressed relax less to NO . Oxidative inactivation of target proteins for NO can be associated with the pathogenesis of cardiovascular diseases. Oxidative inactivation of SERCA is also implicated in dysregulation of smooth muscle migration, promotion of platelet aggregation and impairment of cardiac function, which can be associated with restenosis, pathological angiogenesis, thrombosis, as well as heart failure. Specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295 were found also in myocyte contractile dysfunction in the mouse heart mediated by reactive oxygen species  irreversibly oxidizes Cys674 or nitrate tyrosine residues at Tyr 296-Tyr297, which is associated with loss of SERCA function  and atherosclerosis  sites observed both in aging and in ischemia/reperfusion were found in cardiac and skeletal muscles, while conformational changes in the transmembrane domain of SERCA were associated with adjuvant arthritis. Several authors concluded that SERCA oxidative posttranslational modifications contributed to the pathophysiology of individual diseases and that preservation of SERCA function and/or increasing expression of SERCA may be a novel strategy against diseases associated with oxidative stress  dramatically improved the smooth muscle response to NO. The beneficial effects of the antioxidant depended on improvement of the response to NO in the smooth muscle cells (SMC) .In the streptozotocin-induced diabetic rat model, elevated plasma angiotensin II levels were improved by treatment with the angiotensin-converting enzyme inhibitor enalapril. As angiotensin II is a powerful inducer of NADPH oxidase, it is likely that enalapril helps prevent the diabetes-related impairment of SERCA function in the aorta by suppressing ROS formation .The enhanced tyrosine nitration and inactivation of SERCA2 in platelets from type 2 diabetic patients were restored by treating the patients with rosiglitazone. This was accompanied by improvements in platelet i and at least partially via sarcoplasmic/endoplasmic reticulum Ca2+-ATPases. Thus flavonoids may be auseful tool for preventing cancer development.Flavonoids are able to initiate apoptosis, especially in cancer cells, in vitro and in vivo induced by ROS/RNS, the changes in calcium transport can further lead to tissue injury, cell killing and impaired cardiac contraction. Some flavonoids significantly increased cell viability and protected against cardiomyocyte apoptosis, via preservation of SERCA function, which can be a novel strategy against cardiovascular diseases associated with oxidative stress.On the other hand, decrease of apoptosis is required in some cases. In cardiac hypertrophy


Intracellular Ca2+ concentration ([Ca2+]i) is regulated by two flux pathways; Ca2+ oscillations evoked by the release of Ca2+ from the endoplasmic reticulum, and/or Ca2+ entry from the extracellular fluid. The latter is carried out by the plasmamembrane localized Ca2+ permeable channel such as "transient receptor potentials (Trps)". Trpv4-deficient mice show an increased bone mass due to impaired osteoclast maturation, because Trpv4 mediates Ca2+ influx at the late stage of osteoclast differentiation and hereby regulates Ca2+ signaling [2+ transport [R616Q/V620I) and characterized it on Ca2+ signaling especially in the occurrences of oscillations at the initial step of osteoclast differentiation.Osteoclast differentiation is critically dependent on cellular calcium  and 3 fold in Trpv4R616Q/V620I (p < 0.01) compared to controls. Although spontaneous Ca2+ oscillations were absent in control progenitor cells, Trpv4R616Q/V620I progenitor cells already displayed irregular oscillatory pattern.Intact Trpv4 and Trpv42+ permeable channel supports Ca2+ oscillations in progenitor cells and therefore promotes the potential of osteoclast differentiation.In summary, our findings provide evidences that the activation of Ca


Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca ATP channels make membrane depolarization by an injected current highly inefficient i) in beta cells stimulated by glucose showed that the bursts of electrical activity have the same frequency, are in phase and of similar shape not only in pairs of cells lying close together [2+]i i and membrane potential oscillations at the level of many beta cells is only circumstantial and it remains unclear (i) to what extent the electrical and calcium signals are temporally synchronized, (ii) whether detectable waves of membrane potential oscillations spread across islets, and (iii) whether they do so in the same direction and with a comparable velocity as waves of [Ca2+]i changes. Therefore, the evidence for waves of membrane potential oscillations and the extent of coupling between [Ca2+]i) of a large number of cells in an islet´s cross-section [2+]i dynamics into more detail, we also performed whole-cell patch-clamp experiments, [Ca2+]i imaging experiments and finally double staining of tissue slices with both voltage- and calcium-sensitive dyes. To address these questions that are fundamental to our understanding of the beta cell syncytium as well as other comparable syncytia, we combined the tissue slice method  with liv-section ,23. ThisThe study was conducted in strict accordance with all national and European recommendations pertaining to work with experimental animals, and all efforts were made to minimize suffering of animals. The protocol was approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection (permit number: 34401-61-2009/2). 3, 6 glucose, 6 lactic acid, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 0.5 ascorbic acid) at 40°C was injected into the proximal common bile duct clamped at the papilla of Vater. Immediately thereafter, the pancreas was cooled by pouring over it an ice-cold ECS. Then the agarose-injected pancreas was extracted and gently washed in ice-cold ECS, small blocks of tissue (0.1-0.2 cm3 in size) were cut and finally transferred to a 5 ml Petri dish filled with agarose at 40°C. Individual cubes of cooled agarose containing tissue blocks were cut from hardened agarose and glued  onto the sample plate of the VT 1000 S vibratome . The tissue was cut at 0.05 mm s-1 and 70 Hz into 140 µm-thick slices of a surface area of 20-100 mm2. Throughout preparation and during slicing the tissue was held in an ice-cold ECS continuously bubbled with a gas mixture containing 95% O2 and 5% CO2 at barometric pressure to ensure oxygenation and a pH of 7.4. After cutting the slices were collected in 30 ml of HEPES-buffered saline at room temperature  150 NaCl, 10 HEPES, 6 glucose, 5 KCl, 2 CaCl2, 1 MgCl2; titrated to pH=7.4 using 1 M NaOH) and then incubated in the dye-loading solution. All chemicals were obtained from Sigma-Aldrich  unless indicated. Tissue slices were cut from pancreata of 10-20 week old NMRI mice of either sex as described in detail previously . In brie2+]i imaging 10-12 slices were incubated in a Petri dish (5 ml) filled with 3.333 ml of HBS containing 6 μM Oregon Green 488 BAPTA-1 AM calcium dye , 0.03% Pluronic F-127 (w/v) and 0.12% dimethylsulphoxide  for 50 minutes on an orbital shaker (50 turns min-1) at room temperature and protected from light. Imaging was made within 12 hours after staining. Following staining, slices were kept protected from light in a dye-free HBS which was exchanged every two hours. Fluorescence intensity changes were analyzed and presented as described previously i oscillations, up to 4 slices were incubated in a Petri dish (5 ml) filled with 3 ml of HBS containing 1.3 μM VoltageFluor (VF2.1) dye and 7.4 μM Rhod-2 AM calcium dye , 0.12% Pluronic F-127 (w/v) and 0.55% DMSO (v/v) for 45 minutes on an orbital shaker (50 turns min-1) at room temperature and protected from light. Rhod-2 was preferred in the double staining experiments due to its spectral properties that enabled discrimination of its signal from the VF fluorescent signal  mounted on an upright Nikon Eclipse E600 FN microscope . OGB-1 was excited at 488 nm and VF at 500 nm with a monochromator . Monochromatic light was reflected by a dichroic mirror (405 nm) and directed through a 60x water immersion objective (NA = 1.0). The emitted fluorescence was transmitted by the dichroic mirror and further filtered through a 520-nm long pass filter. Images (256 x 256 pixels) were taken at a frequency of 2 Hz (light exposure time 100 ms per image) for the [Ca2+]i and at a frequency of 5 Hz (light exposure time 100 ms per image) for the VF. For the measurement of [Ca2+]i imaging using OGB-1 was performed on a Leica TCS SP5 AOBS Tandem II upright confocal system using a Leica HCX APO L 20x water immersion objective (NA = 1.0). OGB-1 was excited by an argon 488 nm laser and the emitted fluorescence was detected by Leica HyD detector in the range of 500–700 nm . Individual slices were imaged in a temperature-controlled bath chamber mounted on the microscope  continuously perifused with ECS. Sampling rate was 20-50 Hz at 256 x 64 pixels.Confocal [Ca2+]i were performed on a Leica TCS SP5 II inverted confocal system using a Leica HCX PL APO CS 20x immersion objective (NA = 0.7). VF and Rhod-2 were excited by an argon 514 nm and a diode pumped solid state (DPSS) 561 nm lasers, respectively. Fluorescence was detected by Leica HyD detectors  in the range of 520-554 nm for VF and 570-700 nm for Rhod-2. In the bath chamber, the slice was continuously perifused with the bubbled ECS at 35 -37 °C. Sampling rate was 5-172 Hz at either 512 x 256 pixels or 512 x 128 pixels (binned 2x2 or 4x4 for higher frequency sampling). To confirm that VF followed the voltage changes in beta cells, we performed a control experiment measuring membrane potential from the whole-cell patch-clamped cell while simultaneously measuring VF signal from the neighboring cells  on one hand and the need to keep the optical sections as thin as possible. Before and after recording a time series, a high spatial resolution fluorescence image was taken and used as a reference to assess motion artifacts and regions of interest (ROIs) for the off-line analyses of dynamics.+-based solution. Fast pipette capacitance (Cfast), slow membrane capacitance (Cslow) and series conductance (Gs) were compensated accordingly. Only experiments with Gs>50 nS were analyzed. Recordings were performed in the standard whole-cell mode via a patch-clamp lock-in amplifier  connected to a PC via A/D converter  and recorded on the PC hard disk using WinFluor V3.4.1 software  at a sampling rate of 1 kHz. The same software was used to apply voltage protocol for identifying beta cells by their Na+ current inactivation properties i oscillations were analyzed off-line. ROIs were selected as indicated in 2+]i detection cell interior only, as indicated in t/F0t - 1, Ft being the fluorescence signal recorded at an individual time point during the experiment and F0t being the basal fluorescence signal at an individual time point estimated from the fit, respectively. Membrane potential and i oscillation onset, recorded with the CCD camera, was determined as a deflection of the signal from the base line. Figures were plotted using the Matlab, SigmaPlot v11.0  and CorelDRAW X5 (Corel Inc). Membrane potential traces were smoothed with a moving average filter with local regressions calculated using weighted linear least squares and a 22+]I oscillations are of similar shape and second, there was no systematic correlation between the intensity of calcium indicator loading and the order of activation of different cells.Heterogeneous loading of cells with the calcium and membrane potential indicators could theoretically introduce artificial heterogeneity in the calcium and membrane potential responses between different cells. We believe that this was not of crucial importance in our study for two reasons. First, it is evident from the presented traces that qualitatively, the temporal profiles of membrane potential and [Ca2+]i) oscillations in the pancreatic tissue slices, previously described in isolated islets [2+]i oscillations in responsive beta cells from a cross-sectional plane of an islet with a CCD camera and the membrane potential oscillations of a single beta cell of the same islet with the whole-cell patch-clamp technique. A single glucose stimulation protocol was used in this and other experiments. Extracellular solution containing 6 mM glucose was used as non-stimulatory condition, whilst raising the glucose concentration to 12 mM served to stimulate beta cells. Upon stimulation, beta cells responded in a characteristic way that allowed for their functional discrimination, with a transient period of continuous bursting accompanied by a transient increase in [Ca2+]i, followed by intermittent bursting accompanied by in-phase [Ca2+]i oscillations superimposed on a sustained plateau of [Ca2+]i, as described previously [2+]i oscillated at regular intervals (7 oscillations/min) . The membrane potential oscillation recorded from a cell positioned approximately in the middle of the cross sectional area displayed typical bursts (slow oscillations with superimposed spikes) of electrical activity with a frequency identical to the frequency of [Ca2+]i oscillations recorded in the surrounding cells. By comparing the timing of the first spike in the patch-clamped cell with onsets of [Ca2+]i oscillations  , with onngerhans . In accothe cell . In Figullations , we foun2+]i and membrane potential oscillations and waves, and to finally determine which of the two waves temporally precedes the other, we resorted to confocal laser scanning imaging using calcium- and voltage-sensitive dyes. To confirm the latter assumption, to compare the duration and velocities of [Ca2+]i waves employing confocal imaging of OGB-1 loaded acute pancreas tissue slices [2+]i that were spreading over the optical plane of the islet of Langerhans as described previously  and end (end50) of a given oscillation. Since waves of [Ca2+]i oscillations often did not follow a linear route, we determined the velocity of the waves by calculating vectorial trajectories describing the average wave paths for the starts50 and ends50 of a [Ca2+]i oscillation  and end50 (red) of the membrane potential oscillations and the directions of the start50 trajectories aligned to 0°. In two out of six islets used for this purpose, the average direction of end50 of membrane potential oscillation fell within 90° of the average direction of start50 of membrane potential oscillation, in two islets end50 of membrane potential oscillation progressed in roughly the opposite direction as the start50 of membrane potential oscillation (angle > 180°), whereas the remaining two islets displayed intermediate angles of end50 of membrane potential oscillation. Next, we assessed the properties of membrane potential oscillations and spreading of membrane potential oscillations at the level of the beta cell functional syncytium in the pancreas tissue slice. This was achieved by using a novel voltage-sensitive dye VoltageFluor (VF) with an enhanced linear fluorescence response per voltage change  that enahe frame . The sam2+]i, and by the patch-clamp experiments are shown and compared in 2+]i oscillation spreading determined by confocal imaging are shown and compared in 50 and start50) assessed by the VF dye was 2.4 s . This value is not statistically significantly different from the median duration of oscillations measured with the patch pipette at a much higher temporal resolution, i.e. 1 kHz . Additionally, comparing membrane potential oscillation durations with the [Ca2+]i oscillations also showed no statistically significant difference . Finally, the velocities at which the membrane potential and [Ca2+]i oscillations were spreading were not statistically significantly different . Durations of oscillations in different islets, as determined separately by the confocal imaging of the membrane potential, the [Caifferent   at concentrations above 5 mM replaces the characteristic bursting pattern (slow waves with superimposed fast spikes) with continuous spiking consisting of much taller and longer spikes with no underlying slow waves [2+]i kinetics. Additionally, since the currently achievable combination of imaging speed and voltage indicator properties did not enable us to resolve individual fast spikes superimposed on slow waves during stimulation with glucose, stimulation with TEA in addition to glucose enabled us to characterize the taller and longer spikes present during stimulation with stimulatory glucose plus TEA and their degree of synchronization in different cells. 2+]i oscillations in islets of Langerhans during stimulation with 12 mM glucose plus 10 mM TEA. We used the generic term oscillations to describe the membrane potential changes detectable by VF and produced by slow waves with superimposed spikes in case of stimulation with glucose only, as well as to describe the membrane potential changes detectable by VF and produced by large amplitude regular spikes with no underlying slow oscillations in case of stimulation with glucose plus TEA. Similarly to stimulation with glucose only, both membrane potential and [Ca2+]i oscillations spread across the focal plane in a wave-like manner. However, the [Ca2+]i signal differed markedly from the membrane potential signal in terms of its kinetics, the [Ca2+]i return to the baseline being markedly prolonged compared with the membrane potential signal, which is reflected in significantly longer durations of [Ca2+]i oscillations compared with the membrane potential oscillations . Interestingly, TEA also increased the speed at which the membrane potential and [Ca2+]i waves spread over the islet by almost ten-fold . For comparison of velocities at which the membrane potential and the [Ca2+]i wave spread in islets during stimulation with glucose with and without TEA see also For the above oscillation durations, the rather large variability between cells from different islets could mask any potential systematic differences between durations of the membrane potential and i reporter dyes during stimulation with 12 mM glucose . The described time delay is clearly visible in time traces in 2+]i. The median time lag between end50 of the membrane potential and [Ca2+]i oscillations was 360 ms .To unambiguously resolve whether any of the two signals precedes the other at the level of individual cells, we took up the task of simultaneously staining beta cells with calcium- and voltage-sensitive dyes. We successfully recorded from two islets the simultaneous response of the membrane potential and [Ca glucose . VF load2+]i oscillations at the level of individual cells changes when 12 mM glucose plus 10 mM TEA are used for stimulation. To clarify this, we double-stained four islets with the membrane potential and [Ca2+]i reporter dyes and recorded their responses during stimulation. With this protocol, the differences in the kinetics of both signals became even more obvious. The VF signal showed a very fast upstroke and return to the baseline, whereas the Rhod-2 signal showed slower upstroke of the signal and its return to the baseline was slower by several tens of milliseconds. The time lag between the start50 of the membrane potential and of the [Ca2+]i oscillation was significantly decreased compared with oscillations in glucose only . Conversely, the time lag for the ends50 was decreased to a smaller extent  compared with conditions without TEA.. For comparison of parameters during stimulation with glucose and glucose plus TEA, see Finally, we wanted to explore how coupling between membrane potential and [Ca2+]i oscillations using the patch-clamp technique and CCD camera imaging has been previously attempted on isolated islets [2+]i oscillation as both processes seem time locked. In our hands, 100 ms exposure of the sample to the fluorescent light has been sufficient to image the OGB-1 signal; however, using this exposure period with a frequency higher than 3Hz completely abolished the membrane potential oscillatory activity in the patch-clamped cell thus preventing higher temporal resolution recordings using the CCD camera. Finally, the classical electrophysiological approach of recording membrane potential has the inherent limitation that only a few cells at most can be recorded per islet in parallel. This strongly limits the amount of available data that would reliably describe the electrical network of beta cells and therefore describe the coupling of the membrane potential and [Ca2+]i on a population level. A qualitative description of the extent of coupling between membrane potential and i dynamics, we did not further investigate this issue.The VF signal recorded from islets of Langerhans in acute pancreas tissue slices during stimulation with glucose showed the characteristic oscillatory pattern, very similar in period to the slower carrying component of the one observed with the patch pipette (i.e. bursts) during the plateau phase of the response to 12 mM glucose  and 3. H50 of oscillations formed a specific spatio-temporal activation map that for the first time clearly revealed a wave-like spreading of depolarization over the optical plane of the islet. Comparing properties of membrane potential dynamics with the ones of [Ca2+]i dynamics showed that membrane potential oscillations were of shorter duration than [Ca2+]i oscillations during stimulation with 12 mM glucose at the single cell level. The durations of membrane potential and [Ca2+]i oscillations assessed non-simultaneously in different islets did not differ from the durations of bursts, assessed by using the patch pipette, a method that has a several-fold higher temporal resolution compared with imaging approaches. The median velocity at which the depolarization spread over the islets was 69 µm/s, a value very similar to the velocity of [Ca2+]i oscillations measured in tissue slices in this study  to a stimulatory concentration of glucose replaces the bursting pattern (i.e. slow waves with superimposed spikes) with continuous spiking consisting of much taller and longer spikes only (i.e. with no underlying slow waves) that reach from the threshold of slow waves to positive values and are of approximately double the amplitude than slow waves with superimposed spikes combined. The duration of these spikes is an order of magnitude shorter than the duration of slow waves with superimposed spikes  but several times longer than the duration of individual spikes superimposed on the slow waves [2+]i dynamics under this conditions and to see whether waves would still be detectable and their velocity of spreading would increase. The physiological background underlying the effect of TEA is beyond the scope of this study i is expected to follow the membrane potential change more rapidly. In addition to the several-fold increased velocity of the membrane potential and [Ca2+]i oscillation spread established already in the single-staining experiments, our double-staining experiments revealed that during stimulation with glucose plus TEA, detectable time lags between the onsets of the membrane potential and [Ca2+]i oscillations indeed decreased at the employed sampling frequency. In contrast, upon repolarization, the delay in [Ca2+]i return to baseline was decreased to smaller extent under these conditions indicating that the decrease in [Ca2+]i upon termination of electrical activity is limited by the activity of the [Ca2+]i handling machinery. To test our hypothesis that the rate of initial depolarization determines when [Ca2+]i oscillations, while the velocity at which depolarization spread over the islet was comparable to the one for [Ca2+]i waves. For beta cells in their native environment, we reported for the first time simultaneous records of membrane potential and [Ca2+]i oscillations during stimulation with glucose at cellular resolution. The membrane potential oscillations were phase-locked to the [Ca2+]i oscillations, with the former preceding the later by > 100 ms at the oscillation starts and by > 300 ms at the oscillation ends. The potassium channel blocker tetraethylammonium (TEA) increased the spreading velocity of both membrane potential and [Ca2+]i oscillations by several-fold and emphasized differences in the kinetics of membrane potential and [Ca2+]i changes. The delay between the membrane potential and [Ca2+]i oscillations was decreased to < 50 ms at start50 and to < 250 ms at ends50. Observed kinetic differences suggest that an in-depth analysis of the electrotonic spread between individual beta cells and of molecular determinants of intracellular calcium homeostasis has the potential to further elucidate the function of the beta cell syncytium in health and disease.In the present paper, we demonstrated that during stimulation with glucose oscillatory membrane potential changes spread over the islet in a wave-like manner. The duration of membrane potential oscillations was shown to be shorter than the duration of [Ca2+]i signaling are coupled at the level of individual cells and how they are integrated at the level of an islet, enabling individual cells to function as a unit. In our opinion, combining voltage- and calcium-sensitive dyes with high resolution confocal imaging in acute tissue slices provides a powerful tool for studying the beta cell syncytium and similar cellular syncytia. Our work upgrades our understanding of how membrane potential and [CaFigure S1Membrane potential oscillations upon stimulation with 12 mM glucose.A Fluorescence trace of the voltage-sensitive dye VF measured with confocal imaging. Cell location within islet of Langerhans is marked in B. Numbers denote glucose concentration in mM. B Cell membranes in a representative islet of Langerhans are labeled with the voltage-sensitive dye VF. C Membrane potential oscillations indicated in panel A with the gray rectangular area are shown in detail. Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. Sampling rate 1Hz at 512 x 256 pixels (from which 246 x 256 frame is shown).(TIF)Click here for additional data file.Figure S2i and membrane potential waves during stimulation with 12 mM glucose and 12 mM glucose plus 10 mM TEA.Velocities of [Ca] Distance vs. time is plotted for the calculated trajectories during stimulation with 12 mM glucose (blue) and 12 mM glucose plus 10 mM TEA (red), assessed using the VF (circles) and the OGB-1 (diamonds) dye. The respective linear regression lines are drawn for the VF (solid line) and OGB-1 (dashed line) data during stimulation with glucose only (blue) and with glucose plus TEA (red). Note that the slopes of the regression lines directly represent the wave velocities: 188 and 106 µm/s for the VF and OGB trajectories during glucose only stimulations  and 769 and 1031 µm/s for the VF and OGB trajectories during glucose plus TEA stimulation . (TIF)Click here for additional data file.Figure S350 and ends50 during stimulation with glucose and glucose plus TEA.Delays between the Rhod-2 and VF signals of startsA During stimulation with 12 mM glucose the delay between starts50 of the Rhod-2 and VF signals  is statistically significantly longer than during stimulation with 12 mM glucose plus 10 mM TEA . Asterisks indicate p<0.001 (Mann Whitney test). B During stimulation with 12 mM glucose the delay between ends50 of the Rhod-2 and VF signals  is statistically significantly longer than during stimulation with 12 mM glucose plus 10 mM TEA . Asterisk indicates p<0.05 (Mann Whitney test). (TIF)Click here for additional data file.Figure S42+]i (using Rhod-2 dye).Experimental setup for simultaneous recording of membrane potential (using VF dye) and i dynamics measured with two different calcium indicators, OGB-1 and Rhod-2.Comparison of [CaA Slices were loaded with a loading mixture containing both OGB-1 AM and Rhod-2 AM. Indicated is a region of interest that is analyzed in C-F. B Excitation (broken line) and emission (solid line) spectra of OGB-1 (green) and Rhod-2 (red). Vertical broken lines indicate the 488 nm laser light exciting both dyes and the 561 nm laser light exciting Rhod-2 only. Detector 1 was set to measure OGB-1 signal only (0.4 % of the Rhod-2 emission is detected) whereas the detector 2 captured mostly Rhod-2 and partly OGB-1 emission . C 12 mM glucose elicited [Ca2+]i oscillations superimposed on the plateau phase of the response. Both detectors measured equal shapes of the seven [Ca2+]i oscillations. Note that the red signal is several times larger than the green signal at equal detector gains. In this and all other panels, signals from detector 1 and 2 are depicted in green and red, respectively. D A detailed representation of a single oscillation from C. [Ca2+]i dynamics of the green and red signals are identical. E The oscillation onset from panel D is shown in detail. For both the green and the red signals the starts50 of [Ca2+]i oscillations were determined at their half-maximal amplitude (indicated by arrows). Original (thin lines) and filtered data (thick lines) are shown. F End of the oscillation from panel D is shown in detail. Ends50 of [Ca2+]i oscillations were determined for the red and green signals at their half-maximal amplitude (indicated by arrows). Original (thin lines) and filtered data (thick lines) are shown. G Differences in starts50 and ends50 of [Ca2+]i oscillations of the red and green signals for the 7 oscillations in 9 cells. The difference between the two signals was not statistically significantly different from zero for starts50 and ends50 (p>0.05). (TIF)Click here for additional data file.Figure S6Simultaneous recording of membrane potential change with the VF dye and with the patch-clamp technique.A The VF dye loaded many cells in this islet of Langerhans. White lines indicate location of the patch-clamp pipette. B A scheme indicating active cells. The patch pipette is in contact with the cell from which the membrane potential was recorded with the whole-cell patch-clamp technique. Its neighboring cells from which the membrane potential was measured as VF fluorescence are marked with numbers. C In the patched-clamped cell the membrane potential depolarization was followed by superimposed spikes (lower trace marked with PATCH Vm). In addition to the raw signal, a smoothed signal of the membrane potential change is shown (thicker solid line). In the neighboring cells the VF fluorescence transiently increased to a new plateau value (traces marked with VF Vm). Note the equal timing and shape of the fluorescence signal compared to the smoothed signal recorded with the patch-clamp. The high frequency spikes are missing from the fluorescence signal due to sampling rate of the VF signal (5 Hz). Sampling rate for the patch-clamp was 1000 Hz. Traces represent the normalized fraction of the difference between maximum and plateau baseline fluorescence. The VF fluorescence was captured using the CCD camera at 256x256 pixels. (TIF)Click here for additional data file.Text S12+].Substantiation of the Rhod-2 as indicator of cytosolic [Ca(DOCX)Click here for additional data file.


In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT These modifications play a significant role in various signaling transductions though affecting the activity and structure of different kinds of enzymes, receptors, and ion channel proteins et al demonstrated that alkylating agent NEM and NO donor 5-amino-3--1,2,3,4-oxatriazolium (GEA3162) just stimulated calcium entry through a non-store-operated pathway via direct sulfhydryl modification et al reported that NEM and membrane-permeant NO donors could activate non-store-operated calcium entry through sulfhydryl modification in DDT1MF-2 cells 3) receptor-sensitive stores via S-nitrosylation of sulfhydryl groups 2+]c of activated neutrophils as compared to resting neutrophils. The present study investigated the calcium entry mechanism induced by the alkylation agent NEM and the exogenous NO donor SNP in PMA-activated human neutrophils. We asked whether the effects of NEM and SNP on [Ca2+]c were due to the activation of neutrophils. Data showed that NEM or SNP-triggered [Ca2+]c increase was due to a IP3 receptor-sensitive calcium release and SOCE via sulfhydryl modification in PMA-induced activation-dependent human neutrophils.For resting rat neutrophils, Wang 2+]c responses to thapsigargin (TG) 2+]c of PMA-pretreated neutrophils. Addition of 2 µM TG induced a sharp rising of [Ca2+]c due to calcium store release in Ca2+-containing medium, followed by a second extended rising of [Ca2+]c  was added during the entry phase, it rapidly and obviously abolished calcium entry  activator, can activate NADPH oxidase by stimulating PKC and subsequently induce respiratory burst in neutrophils  [Ca2+]c . The latum entry . When 75g buffer . It is w2+]c increase followed by a second extended rising of on [Ca2+]c in PMA-preincubated neutrophils within Ca2+-containing HBSS. When cells were pretreated with NEM (500 µM), the effect of SNP on [Ca2+]c was significantly abolished  or SNP (500 µM) in the absence of Ca2+ followed by readdition of external Ca2+ in PMA-activated neutrophils. It was found that subsequent addition of external Ca2+ resulted in a further calcium entry event after stimulation by NEM or SNP, demonstrating calcium entry should be store-depletion-dependent  using spectrofluorometer. It was found that NEM (100 µM) or SNP (500 µM) did not caused [Ca2+]c increase with Ca2+-containing buffer in resting neutrophils as shown in 2+]c in resting neutrophils served as a positive control  caused no increase in [Ca2+]c elevation when cells were preincubated with 150 µM 2-APB as shown in △- curve) and B . This result indicated that calcium release came from IP3 receptor-sensitive stores. Meanwhile, application of 75 µM 2-APB prior to NEM stimulation completely blocked the calcium entry . In addition, addition of 75 µM 2-APB to cells already responding to NEM or SNP resulted in rapid decrease in [Ca2+]c back to basal levels  had little capacity for inducing NEM (100 µM) or SNP (500 µM)-pretreated [Ca2+]c change in PMA-activated human neutrophils (2+]c (The classic G protein-coupled receptors (GPCRs)-G protein-Phospholipase C (PLC)-IPtrophils . This inls -3-methoxyestra-1,3,5,(10)-trien-17-yl]-amino)-hexyl]- 2,5-dione (U73122), a putative inhibitor of PLC 2+]c elevation in Ca2+-containing medium when PMA-activated neutrophils were treated with U73122 (10 µM) as summarized in 0: 5.062±0.121 for NEM vs. 1.110±0.012 for U73122+NEM, n = 30 cells, P<0.01). Meanwhile, the effect of SNP on [Ca2+]c was completely inhibited in Ca2+-containing buffer in the presence of U73122  to achieve an apparent SOCE observed . Besides, much research has demonstrated that 2-APB was a reliable blocker of SOCE in recent years et al reported that 2-APB showed obvious inhibitory effect on SOCE at 75 µM 3 receptor-sensitive stores-operated pathway in PMA-activated human neutrophils.2-APB, a specific inhibitor of IP2+]c increase (data not shown). However, further result showed that neutrophils could not spread on glass evidently when neutrophils were preincubated with 200 nM for 5 min at 37°C. Subsequent application of NEM or SNP had no obvious effect on [Ca2+]c (data not shown). It could be proposed that the activation ability of PMA on neutrophils were concentration and activation time-dependent. Lower concentration of PMA used, more activation time needed. The degree of neutrophils adhesion on glass substrate may have effect on sulfhydryl modification-induced [Ca2+]c change.In addition, it is easy to find that we use a relatively high an order (2 µM) of magnitude higher than usual-PMA-dose to activate neutrophils from the resting state. It should be important to see whether the same phenomenon occurs at a submicromolar concentration. First, we found that 200 nM PMA (30 min at 37°C) could result in neutrophils obvious spreading on glass. Under this condition, NEM (100 µM) or SNP (500 µM) caused obvious c increase when neutrophils was preincubated with PMA just at submicromolar concentration for little time at 37°C (data not shown). These evidence suggested the activation degree of respiratory burst may contribute to generation of sulfhydryl modification-induced SOCE in neutrophils. However, further study is still needed to elucidate the relationship between activation of respiratory burst-generating NADPH oxidase and sulfhydryl modification.Interestingly, Wang 3-sensitive store-operated pathway via sulfhydryl modification in PMA-induced activation-dependent human neutrophils. Since SOCE plays a crucial role in neutrophils, this study on investigating sulfhydryl modification-induced SOCE may help contribute towards understanding mechanisms of [Ca2+]c regulation surrounding neutrophil inflammatory reactions.In summary, we clearly demonstrated that NEM, a thiol-alkylating agent, and SNP, a donor of exogenous NO, induced obvious calcium entry through IPThe study was reviewed and approved by the Ethics Committee of Nankai University. All participants gave written informed consent.SNP, NEM, PMA, FMLP, 2-APB, TG, U73122, DPI, EGTA, Histopaque 1077 and 1119 solutions, Hoechst 33342, trypan blue were obtained from Sigma–Aldrich . Fluo-3 acetoxymethyl ester (Fluo-3 AM) was from Gibco .2, 1 mM MgCl2, 10 mM Hepes and 10 mM glucose, PH adjusted to 7.4 with NaOH 1 mM) and then kept in an ice bath before use.Neutrophils were obtained from the blood of normal healthy adults by step-density gradient centrifugation over Histopaque 1077 and 1119 solutions at 500×g for 15 min. The cells were >96% viable as assessed by trypan blue exclusion and had a purity of >95% as demonstrated by Hoechst 33342 nuclear staining. Cells were suspended in HBSS  were observed microscopically at 37°C by using a temperature-controlled stage. Fluorescence was performed with a fluorescent microscope  with a 100 W mercury lamp. A shutter  was equipped behind the mercury lamp to control the exposure time and the interval time for fluorescence imaging. Fluorescence signaling was detected by an electron multiplying charge coupled device (EMCCD)  which was connected to the left exit side of microscope. The EMCCD was controlled by MetaMorph software version 7.1 . DIC and fluorescence images were collected by this microscopy system.2+]c was measured by the micro-imaging system as shown above. Fluo-3 AM-loaded cells were excited by a mercury lamp with a 485/20 nm excitation filter, and fluorescence was collected by a fluar oil objective with a 510 nm long-pass dichroic mirror and a 540/50 emission filter. Each fluorescence image was acquired for 50 msec with a 2 s interval between frames. The obtained images were quantitatively analyzed for changes of fluorescence intensities within region of interest. [Ca2+]c change was represented by relative fluorescence intensity . At least 30 individual cells were selected from three independent experiments, with one characteristic [Ca2+]c trace plotted to represent >10 similar traces. Besides, Ca2+-free HBSS was prepared by substituting MgCl2 for CaCl2 at the same concentration, with 2 mM EGTA. For [Ca2+]c measurement in suspension, Fluo-3 AM-loaded neutrophils were suspended in HBSS to 1×106cells/mL. Fluorescence was monitored with a spectrofluorometer  at 530/30 nm with excitation at 488 nm.Human neutrophils were incubated with calcium-sensitive probe Fluo-3 AM 4 µM for 30 min at 37°C in HBSS. For single cell calcium imaging, [Cat-test. P<0.05 was considered statistically significant.All data were presented as means ± standard error of the mean (S.E.M.). The statistical comparison between groups was carried out using Student’s


The central role of Ca2+ in cellular physiology of neurons is based on a Ca2+-signaling toolkit that assembles intracellular signaling systems with different spatial and temporal dynamics  and by transport of the ion across plasma and intracellular membranes. The complex regulation of cytosolic Ca2+ concentrations is the subject of an increasing number of investigations, because this regulation is intimately linked to the function of Ca2+ in neurotransmitter release, synaptic plasticity, neurosensory signaling, activity-dependent gene transcription, intracellular trafficking, and many other cellular processes. The multitude of Ca2+-regulated processes requires specialized downstream processing machinery, translating the Ca2+ signal into alterations of cellular functions. It is generally believed that the versatile basis for the complex signaling in micro- and nanodomains of neuronal subcompartments is provided by the existence of a multitude of different CaBPs from which numerous belong to the EF-hand super-family. EF-hand proteins are traditionally subdivided into Ca2+ buffer and Ca2+ sensor proteins. This distinction is however not really valid, because Ca2+-binding to EF-hand proteins can serve both functions, even at the same time. Nonetheless, whereas the first group is characterized by a rather high affinity for Ca2+, exhibits little conformational change upon Ca2+-binding and is thought to mainly chelate Ca2+, the second group has a somewhat lower affinity for Ca2+ (often in the 1–10 μM range) and shows considerable conformational changes upon Ca2+-binding, which usually triggers a target interaction.Neuronal Ca2+ signaling toolkit. The general idea of this special issue was to provide an overview on the function of neuronal EF-hand CaBPs in health and disease. The issue contains reviews that summarize the state-of-the-art in the field, as well as experimental and theoretical papers dealing with emerging concepts on Ca2+-signaling/buffering mediated by EF-hand CaBPs. Questions like which features define the functional role of a EF-hand Ca2+ sensor in neurons, the conditions under which a given interaction of a CaBP with its target is of physiological relevance, the emerging synaptic role of these proteins, and mounting evidence for their role in the regulation of protein trafficking are covered. Structural aspects and biophysical studies are included and provocative new ideas based on numerical modeling are part of this issue. Another interesting aspect covered in the research topic is the emerging role of CaBPs in brain disease states. Several papers have shown that CaBPs are targets of drugs in clinical use, that expression levels of CaBPs are frequently altered in brain disease states, and more recently reports on mutations in EF-hand Ca2+ sensors linked to human disease were published. We want to thank all authors for the high quality of the papers that they have submitted and the efforts that they have made to provide an excellent overview of this interesting field. It was a pleasure to edit this research topic.Members of the latter group belong either to the Neuronal calcium sensor (NCS) proteins or the related Caldendrin/CaBP/Calneuron (nCaBPs) family. All of these proteins resemble to a varying degree to the structure of their common ancestor Calmodulin (CaM), but they are quite diverse in amino acid sequence in comparison to CaM. It is therefore surprising that relatively few binding partners for NCS/nCaBP proteins have been identified that are not also CaM targets and this raises the question on the specificity and function of these interactions. Interestingly, binding of target proteins to NCS proteins and nCaBP has frequently different consequences than binding to CaM, which substantially increases the versatility of the Ca


Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca2+-binding to both recoverin and NCS-1 cause large protein conformational changes that ejects the covalently attached myristoyl group into the solvent exterior and promotes membrane targeting (Ca2+-myristoyl switch). The GCAP proteins undergo much smaller Ca2+-induced conformational changes and do not possess a Ca2+-myristoyl switch. Recent structures of GCAP1 in both its activator and Ca2+-bound inhibitory states will be discussed to understand structural determinants that control their Ca2+-dependent activation of retinal guanylyl cyclases.Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca Recoverin  are expressed in other tissues beside the brain  and in lchannels  and actichannels .2+, and a myristoylation consensus sequence at the N-terminus. NCS proteins have similar sequences, ranging from 35 to 60% identity . EF-hand residues are the most highly conserved, particularly in the Ca2+ binding loops. The fourth EF-hand sequence is variable, and Ca2+ is able to bind to EF4 in frequenin . For NCS-1, the attached myristoyl group is located in a protein crevice formed by helices from EF3 and EF4 near the C-terminus . The covalently attached fatty acyl group in NCS-1 protrudes in a parallel fashion between four helices from EF3 and EF4 . The C-terminal location of the myristoyl binding site in NCS-1 is quite different from that of recoverin in which the myristate is positioned inside a cavity near the N-terminus . The attached fatty acyl chain in recoverin is wedged between the helices of EF1 and EF2 in a perpendicular fashion , which contrasts with the parallel arrangement of the fatty acyl chain in NCS-1 . For GCAP1, the myristoyl group is sequestered in a cavity formed by the N-terminal domain with participation of a C-terminal helix . The myristoyl group in GCAP1 bridges the N-terminal and C-terminal ends of the protein by contacting helices at each end . In short, the protein structural environment around the myristoyl group is quite different in recoverin, GCAP1 and NCS-1 . This suggests that each NCS protein folds differently around the N-terminal myristoyl group by contacting non-conserved patches of hydrophobic residues that are unique to each NCS protein. However, myristoylation of GCAP2 is not essential for its ability to activate its target , because unmyristoylated GCAP2 can activate cyclase activity nearly as well as myristoylated GCAP2  and only in the presence of physiological Mg2+ levels  forms a molten globule-like structure, that contains regular secondary structure  is not suitable for ligating either Ca2+ or Mg2+. Ca2+ binds to GCAP1 at the other three EF-hands  in an independent fashion  inhibit RetGC in dark-adapted rods  does not bind to either Ca fashion  in contrecoverin . The appar range , whereasaffinity  in the saffinity . These b–500 nM [ and [Mg2+] ~1 mM ]. Light- 5–50 nM  while thee ~1 mM . Therefosed rods , whereasted rods .2+ binding (but do not affect Mg2+ binding) cause GCAP1 to constitutively activate RetGC in rods and cones, which is genetically linked to various retinal diseases  are unable to inhibit RetGC at high Ca2+ levels in the dark (2+ is bound to EF2 and EF3 (but not bound to EF4). Also, Ca2+ binding to EF4 is essential for having Ca2+-induced inhibition of RetGC. Therefore, Ca2+ binding to EF4 is critical for controlling whether GCAP1 can activate or inhibit RetGC.Mutagenesis studies of the individual EF-hands in GCAP1 have revealed that Caof RetGC . Mutants4N/D148G , 2001] athe dark . In summ2+ binding to GCAP1 and GCAP2 does not cause ejection of the covalently attached myristoyl group  are grouped into two globular domains: N-domain includes EF1 and EF2 (residues 18–83) and C-domain contains EF3 and EF4 (residues 88–161). Ca2+ is bound to GCAP1 at EF2, EF3 and EF4, and the structure of each Ca2+-bound EF-hand in GCAP1  adopts the familiar open conformation of EF-hands as seen in calmodulin and other Ca2+-bound EF-hand proteins . A unique structural feature of GCAP1 is that the N-terminal α-helix (residues 5–15) upstream of EF1 and C-terminal helix (residues 175–183) downstream of EF4 are held closely together by their mutual interaction with the N-terminal myristoyl group . Thus, the covalently attached myristoyl group in GCAP1 is sequestered within a unique environment inside the Ca2+-bound protein, which is quite different from that of recoverin as described above in Figure 2. The myristoyl group attached to GCAP1 makes contacts with N-terminal residues  and the C-terminal helix . In essence, the myristoyl group serves to bridge both the N-terminal and C-terminal ends of the protein, which explains how Ca2+-induced conformational changes in the C-terminal domain (particularly in EF4) might be transmitted to a possible target binding site in EF1. A Ca2+-myristoyl tug mechanism  can activate RetGC at high Ca2+ concentrations (2+ bound at EF2 and EF3) serves as a model of the Ca2+-free/Mg2+-bound activator state. The EF4mut protein is more soluble and stable than Ca2+-free/Mg2+-bound wild type, and EF4mut exhibits NMR spectra with much higher resolution and sensitivity compared to that of Ca2+-free/Mg2+-bound wild type . The overall structure of EF4mut is similar to the crystal structure of Ca2+-bound GCAP1 (root mean squared deviation of main chain atoms is 1.3 Å when comparing the two structures). However, residues at the domain interface (between EF2 and EF3) are structured somewhat differently in EF4mut compared to the crystal structure of Ca2+-bound GCAP1. Many of the GCAP1 residues at the domain interface have quite broad NMR resonances, suggesting that these residues are conformationally dynamic  also exhibited broad NMR resonances and 15N NMR relaxation dispersion studies reveal that these domain interface residues exhibit millisecond exchange kinetics . The most noteworthy Ca2+-induced structural difference in GCAP1 can be seen in the entering helix of EF3 that unravels a half turn in EF4mut, which causes a repositioning of the W94 side-chain at the domain interface. A Ca2+-induced change in the structural environment around W94 is consistent with previous tryptophan fluorescence and electron paramagnetic resonance studies of GCAP1  plays a role in modulating Ca2+-dependent contacts with RetGC1.A GCAP1 mutant, D144N/D148G called EF4mut,  that bintrations . Therefoild type , 2013. N dynamic . The corkinetics . Ca2+-in domains . A strucof GCAP1 , 2007. WFigure 4). GCAP1 residues in EF1 . In our model , Ca2+-induced conformational changes in EF4  by a Ca2+-myristoyl tug mechanism as described by , EF2 and EF3 are bound to Mg2+  with EF1 and EF4 unoccupied. The Ca2+-free state of EF4 forms a loose and dynamic structure  and thus indirectly form hydrophobic contacts with residues in EF1 and EF2. In essence, the myristoyl group forms a bridge between Ca2+-induced conformational changes in the C-terminal domain and the cyclase binding site in the N-terminal domain. In the GCAP1 activator state, residues in the cyclase binding site  are spa and A78 ], which 2+-myristoyl switch that promotes their Ca2+-induced membrane targeting. By contrast, GCAP1 contains a sequestered myristoyl group in both its Ca2+-free and Ca2+-bound states and undergoes rather small Ca2+-induced protein conformational changes. Ca2+ binding to the fourth EF-hand in GCAP1 triggers conformational changes in the N-terminal domain via a Ca2+-myristoyl tug mechanism that controls the exposure of residues in EF1 and EF2 that we propose may serve as a target binding site for guanylyl cyclase.N-terminal myristoylation serves to remodel the structure of NCS proteins as seen for recoverin, GCAP1 and NCS-1. Each NCS protein folds differently around the attached myristoyl group, which causes each NCS protein to adopt a unique structure. Most NCS proteins possess a functional CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.