By impairing release from ribbons, we found that unlike cones where release occurs entirely at ribbon-style active zones, slow release from rods occurs mostly at ectopic, non-ribbon sites. To investigate the role of CICR in ribbon and non-ribbon release from rods, we used total internal reflection fluorescence microscopy as a tool for visualizing terminals of isolated rods loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine. We found that rather than simply facilitating release, activation of CICR by ryanodine triggered release directly in rods, independent of plasma membrane Ca2+ channel activation. Ryanodine-evoked release occurred mostly at non-ribbon sites and release evoked by sustained depolarization at non-ribbon sites was mostly due to CICR. Unlike release at ribbon-style active zones, non-ribbon release did not occur at fixed locations. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that ER extends continuously from synapse to soma. Release of Ca2+ from terminal ER by lengthy depolarization did not significantly deplete Ca2+ from ER in the perikaryon. Collectively, these results indicate that CICR-triggered release at non-ribbon sites is a major mechanism for maintaining vesicle release from rods and that CICR in terminals may be sustained by diffusion of Ca2+ through ER from other parts of the cell.Rods are capable of greater slow release than cones contributing to overall slower release kinetics. Slow release in rods involves Ca Light-evoked voltage changes in photoreceptor cells are transmitted to second-order retinal neurons by changing the rate of glutamate release. In cones, glutamate release is thought to occur almost exclusively at plate-like synaptic ribbons  where deAlthough rods are also capable of fast release , rods ex2+-induced Ca2+ release  from perikaryon to synapse.In the present study, we tested the hypothesis that slow release from rods involves CICR-triggered release of vesicles at non-ribbon sites. To test this hypothesis, we combined electrophysiological recordings and TIRFM visualization of submembrane Can events . The resAmbystoma tigrinum, 18–25 cm in length; Charles Sullivan Co., Nashville, TN, USA) were maintained on a 12-h light/dark cycle and killed 1–2 h after the beginning of subjective night. Salamanders were decapitated with heavy shears, the head was hemisected and the spinal cord pithed. Protocols were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee.Aquatic tiger salamanders , and 5 glucose (pH 7.8). Rods and horizontal cells were simultaneously voltage clamped using Optopatch (Cairn Research) and Axopatch 200B (Molecular Devices) patch-clamp amplifiers. Recording pipettes were pulled on a PP-830 vertical puller  from borosilicate glass pipettes . Pipette resistance was 12–18 MΩ. Rod pipettes were filled with (in mM): 40 cesium glutamate, 50 cesium gluconate, 9.4 tetraethylammonium chloride (TEACl), 3.5 NaCl, 1 MgCl2, 9.4 MgATP, 0.5 GTP, 5 ethylene glycol tetraacetic acid (EGTA), 1 reduced glutathione, 1 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 10 HEPES (pH 7.2). Horizontal cell pipettes contained (in mM): 90 cesium gluconate, 10 TEACl, 1 CaCl2, 3.5 NaCl, 1 MgCl2, 9.4 MgATP, 0.5 GTP, 5 EGTA, 10 HEPES (pH 7.2). Unless otherwise specified, reagents were obtained from Sigma-Aldrich Chemicals . Currents were acquired and analyzed using pClamp 9.2 software with Digidata 1322 interface (Molecular Devices).To measure release from rods electrophysiologically, we obtained paired whole cell recordings from rods and horizontal cells in a retina slice preparation. Details of slice preparation and electrophysiological recordings are described in detail elsewhere . BrieflyTo selectively damage ribbons, we used FALI with a fluorescein-conjugated Ribeye-binding peptide (80 μM) added to the presynaptic patch pipette solution. The Ribeye-binding peptide (EQTVPVDLSVARPR) contains a PXDLS sequence that binds selectively to the C-terminal binding protein (CtBP) domain of Ribeye . After wN-ethylmaleimide-sensitive factor attachment protein receptor) proteins SNAP-23 and SNAP-25, was added to the presynaptic patch pipette solution in some experiments.To block synaptic vesicle release, botulinum toxin E light chain (500 nM), which cleaves the SNARE  to load 1–3% of the vesicle pool  followed by two additional washes in ice-cold, Ca2+-free saline. A piece of retina was then triturated with a fire-polished Pasteur pipette and the cell suspension transferred onto 1.78 refractive index glass cover slips  coated with Cell-Tak . After letting cells settle and adhere for 30 min, they were superfused with oxygenated amphibian saline solution at 20°C. Rods were identified by their characteristic morphology. Cones and sometimes bipolar cells were also loaded with pHrodo but not examined in this study. Light-sensitive outer segments of rods were typically lost during trituration.After loading with pHrodo, retinas were digested by incubation with papain  plus cysteine (0.2 mg/ml) in Ca2+ channels are clustered beneath ribbons  was added to the pipette solution. The sites of peak fluorescence increases evoked by 50-ms depolarizing steps from -70 to -10 mV with ΔF/F > 0.5 were defined as Ca2+ entry sites. Different focal Ca2+ entry sites were defined as separate ribbons if they showed distinct peaks separated by ≥500 nm  for 45 min. Cytoplasmic dye was then washed out by obtaining whole cell recordings with Ca2+- and dye-free pipette solutions . After leaving the objective, light traveled through a high refractive index (1.78) immersion fluid  and entered the cover slip, undergoing total internal reflection at the interface between the glass and lower refractive index of the cell membrane and overlying aqueous medium. The evanescent wave propagated at this interface declined with a length constant of 64 nm with the 561-nm laser and 57 nm with the 488-nm laser  bandpass filters  and images were obtained at 14–31 ms/frame. Imaging data were acquired using Metamorph software  and analyzed with Metamorph and ImageJ 1.46 .561- and 488-nm solid-state lasers  were used to illuminate pHrodo-loaded vesicles and Canm laser . For pHrRods were stimulated by pressure ejection  of 50 mM KCl or 10 μM ryanodine from patch pipettes or by application of depolarizing voltage steps to voltage-clamped rods. The tip of the puffer pipette was positioned 10–20 μm away from rod terminals.For application of depolarizing steps , whole cell recordings were obtained from isolated rods, using the rod pipette solution described above. Rods were voltage-clamped with an A-M Systems Model 2400  amplifier. Currents were acquired and analyzed with pClamp 8 and Digidata 1200 interface (Molecular Devices). Cells with holding currents >300 pA at -70 mV were rejected from analysis.To test whether CICR increased vesicle mobility in rod terminals, we loaded a small fraction of vesicles with pHrodo and then examined rod terminals under epifluorescent illumination. We focused the microscope on the cytoplasm above the membrane to avoid possible effects of release on measured changes in fluorescence. Ryanodine (10 μM) was puffed onto rod terminals to activate CICR. We compared the average frame-to-frame correlations in intraterminal fluorescence among 23 frames in the resting state to the average correlations in the CICR-activated state during the puff.The ER of isolated rods was labeled by incubation with 1 μM ER-tracker green (Invitrogen) for 30 min at 20°C. Terminals of rods were photobleached for 5 s by illumination with a small spot (8 μm diameter) from a 30-mW 488-nm laser. ER-tracker green was illuminated by epifluorescence and images were acquired every 3 s. To show that ER-tracker dye labeled intracellular structures, we also visualized rods using a spinning disk confocal microscope (PerkinElmer Ultraview LCI).t test. The criterion for statistical significance was chosen to be p < 0.05.Statistical analysis was performed with GraphPad Prism 4 . Unless otherwise stated, results are presented as mean ± SEM and statistical significance was determined using Student’s Figure 1A). For FALI, we used a fluorescein-conjugated peptide that binds selectively to the B domain of Ribeye  was reduced by damaging the ribbon but slower components of the EPSC were not. The post-FALI response shown in Figures 1A,B is the third response after bleaching. The first response obtained after FALI was not diminished, consistent with results from bipolar cells and cones indicating that release of vesicles that were previously primed and attached to the ribbon was not impaired by ribbon damage  by the fourth test step after FALI. Bleaching a scrambled control version of the peptide conjugated to fluorescein did not reduce EPSCs .By using capacitance measurements of exocytosis in rods, we found previously that damaging the ribbon protein Ribeye by FALI significantly inhibited release evoked by short 50-ms depolarizing steps but did not significantly inhibit slow release evoked by longer 200-ms depolarizing steps . This suf Ribeye . The pepf Ribeye . Depolarf Ribeye  and addif Ribeye . Consistn damage . As illuFigure 1A). Comparing the charge transfer of control EPSCs to EPSCs after FALI (average of third through sixth responses), EPSCs were reduced by only 2 ± 11% . Assuming that the 49% decline in the amplitude of the fast EPSC component means that half of the ribbon sites were damaged by FALI, this suggests that release from ribbons contributes only ~4% to the total EPSC charge transfer evoked by a 200-ms step to -10 mV.By contrast with the reduction in fast release, damaging the ribbon by FALI did not inhibit slow release . After recording for more than an hour from rod-horizontal cell pairs with botulinum toxin in the presynaptic patch pipette, the total EPSC charge transfer was reduced by 56 ± 14% . The portion that remained (43%) could involve SNARE-independent release  by hyperpolarizing neighboring rods with bright light. These results suggest that 56% of the EPSC was due to release from the voltage-clamped rod. Together with FALI results suggesting that 4% of the EPSC was due to release at ribbons, this suggests that ~7% (4%/56%) of release from voltage-clamped rods evoked by 200-ms steps to -10 mV occurred at ribbons. This supports the conclusion of other studies suggesting that rods are capable of considerable non-ribbon release . Figure 2E shows the increase in fluo-5F fluorescence evoked by ryanodine puff measured within a region of interest in the rod terminal. After emptying ER Ca2+ stores by application of a sarcoplasmic/ER Ca2+-ATPase (SERCA) inhibitor, cyclopiazonic acid , ryanodine failed to stimulate an increase in [Ca2+]i . Ryanodine-evoked CICR was also blocked by pretreatment with another SERCA inhibitor, thapsigargin .Consistent with previous work showing that the machinery for CICR is present in rod terminals , we foun2+ involved in depolarization-evoked increases in submembrane [Ca2+]i. Consistent with earlier findings , reflecting the clustering of Ca2+ channels adjacent to ribbons . Blocking ryanodine receptors by addition of a ryanodine receptor blocker, dantrolene , or a high concentration of ryanodine  to the pipette solution did not affect Ca2+ influx evoked by 50-ms steps , but confined the spread of Ca2+ evoked by 500-ms depolarizing steps as expected from a block of CICR . The time course for Ca2+ changes in two regions of interest , placed over a focal Ca2+ entry site  and away from focal Ca2+ entry sites , further confirmed the effectiveness of the ryanodine receptor-mediated block of CICR during sustained depolarization. After blocking CICR, depolarization with a 500-ms step evoked a localized Ca2+ increase in region 1 near the presumed location of a ribbon but evinced little change in other parts of the terminal . Thus, blocking CICR converted the [Ca2+]i response evoked by a sustained depolarization to a confined response typically observed following brief depolarizing steps.We examined the sources of Cafindings , 50-ms d ribbons . Longer 2+]i throughout the terminal and this spread of Ca2+ was confined by blocking CICR with dantrolene . These findings show that the spread of submembrane Ca2+ during sustained depolarization is due to CICR, consistent with previous studies  increased  increase during 500-ms steps that resembled confined [Ca2+] increases triggered by 50-ms depolarizing steps . Dantrolene also confined release events evoked by 500-ms steps close to focal Ca2+ entry sites . In the presence of dantrolene, most (~75%) of the release events evoked by 500-ms steps occurred within 1 μm of the focal Ca2+ entry sites, matching the distribution of release events evoked by 50-ms steps in the presence or absence of dantrolene . For comparison, salamander rod ribbons average ~1 μm in length  and show that 500-ms steps applied in control conditions evoked a significantly larger number of release events than 50 ms steps  > 1 μm from the ribbon. Consistent with electrophysiological results showing the contribution of CICR to slow release . As illustrated by overlay of a vesicle recruitment map (red) and ribbon locations (green) in Figure 6C, the sites to which vesicles were recruited by activation of CICR did not overlap with ribbons in five out of six rods. In one rod, there was partial overlap. Given that release-ready vesicles are thought to cluster primarily at ribbons, it was surprising to find that direct stimulation of CICR stimulated recruitment and subsequent release almost exclusively at non-ribbon sites. However, the finding that CICR triggers release almost entirely at non-ribbon sites complements the previous finding that non-ribbon release during sustained depolarization is almost entirely due to CICR.To observe the behavior of a large number of vesicles, we incubated rods with pHrodo for 30 min. We mapped sites of vesicle recruitment and subsequent release from increases in near-membrane fluorescence that accompanied membrane approach of vesicles just prior to fusion. Since 85% of near-membrane vesicles subsequently fuse, recruitment maps provide a good map of release sites . CICR waFigure 7A). The clustering of newly recruited vesicles observed during depolarizing stimulation co-localizes with synaptic ribbons  to those evoked by depolarizing stimulation with 50 mM KCl in the same terminals. Recruitment maps were generated in terminals loaded by 30-min incubation with pHrodo as described above. Vesicle recruitment triggered by puff application of 50 mM KCl was concentrated at a few sites in each terminal with some additional recruitment observed elsewhere in the terminal ( ribbons . These r2+ from intracellular stores evoked by puff application of ryanodine (10 μM) stimulated vesicle recruitment and release at more diffusely distributed locations throughout the terminal than high K+ stimulation . Furthermore, sites of vesicle recruitment triggered by activation of CICR with ryanodine differed from sites of vesicle recruitment and release triggered by depolarizing stimulation with 50 mM KCl . Because depolarizing stimulation preferentially stimulated release from ribbon sites, this provides further evidence that CICR preferentially stimulates release at non-ribbon sites.Release of CaFigures 7D–F; n = 7 rods). Vesicle recruitment at these additional sites may be due to activation of CICR by KCl puffs. By contrast with the largely repeatable sites evoked by KCl puffs, two successive puffs of ryanodine triggered vesicle recruitment and release at different sites in the two trials . These results suggest that depolarization-evoked release at ribbons occurs at fixed locations whereas sites of non-ribbon release triggered by CICR are not fixed.To test whether non-ribbon release occurs at fixed locations, we repeatedly stimulated CICR in rod terminals with puff application of ryanodine. We also tested repeated stimulation by puff application of high KCl solutions. To keep cells healthy and improve responses to repeated stimulation, the stimulus strength was reduced . Puff application of KCl twice in succession triggered vesicle recruitment and release concentrated at overlapping locations in both trials although there was also some vesicle recruitment to other sites . Furthermore, evidence that blocking CICR inhibits light responses in second-order neurons and ongoing glutamate release from rods suggests that CICR can be maintained almost indefinitely . To test for continuity of these labeled structures, we measured recovery of terminal fluorescence after photobleaching ER-tracker green with a laser spot  centered on the terminal. We monitored cell fluorescence at 3 s intervals before and after laser photobleaching . By measuring the rate of fluorescence decline in terminals and somas of control cells from the same microscope field that were not photobleached by the laser, we determined that epifluorescent measurements made every 3 s caused a small degree of bleaching , independent of laser photobleach (65%). Epifluorescent measurement caused similar bleaching in terminals  and somas  of control cells. After subtracting the bleaching caused by epifluorescent measurements, the rate of FRAP by the laser exhibited a time constant (τ) of 38.3 s  Without correcting τ for bleaching by the measurement light, terminal fluorescence recovered with τ = 38.8 s . Recovery of terminal fluorescence is due to dye molecules diffusing to the terminal from other parts of the cell, including the soma. We therefore measured fluorescence changes in the somas of cells whose terminals were photobleached. Before correcting for bleaching by the epifluorescent measurement light, the fluorescence intensity declined after laser photobleach more rapidly in somas of cells whose terminals were photobleached  than in somas of control cells whose terminals were not photobleached . After correcting for bleaching by epifluorescent measurement, the fluorescence signal in the soma declined exponentially with τ = 43.1 s, close to the rate of FRAP in the terminal . The amount of fluorescence decline in the soma caused by laser photobleach was much smaller than the amount of fluorescence recovered in the terminal, as expected from the much larger volume of ER in the soma. These results suggest that dye can diffuse from soma to terminal, restoring fluorescence in the terminal and slightly diminishing fluorescence in the soma.Ultrastructural studies of photoreceptors show the presence of ER in the terminal, axon, and soma . In othe another . We hypont on ER . Confoca3R1 receptors on ER membranes is 0.3 μm2/s . We then obtained whole cell patch clamp recordings using a Ca2+- and dye-free pipette solution to wash dye out of the cytoplasm , consistent with that the presence of ER Ca2+ stores close to the membrane. We depolarized rods with 500-ms steps to test whether these bright areas represented functional intracellular Ca2+ stores mediating CICR. As illustrated in Figure 9, ER [Ca2+] decreased in the terminal during the depolarizing step and recovered soon thereafter. This confirms that sustained depolarization releases Ca2+ from intracellular Ca2+ stores in rod terminals. Application of a 500-ms depolarizing step caused only a very small decrease in soma ER [Ca2+]. Similar results were seen in four cells. The decrease in soma ER [Ca2+] could be due to diffusion of Ca2+ through the ER from soma to terminal, although it might also be due to release of Ca2+ into the soma cytoplasm. Regardless, this result shows that release of Ca2+ from terminal ER did not significantly deplete Ca2+ from soma ER during a 500-ms step. Thus, soma ER Ca2+ stores appear to have the capacity to sustain the transfer of Ca2+ from cell body to terminal during sustained depolarization.To visualize ER Ca0–400 μM , we loadytoplasm . Many br2+] to stimulate release from rods, much lower than the [Ca2+] needed to stimulate release from other neurons .Consistent with ultrastructural evidence of ER close to the plasma membrane in rod terminals , activat ribbons , ryanodi buffers . AnotherActivation of CICR by repeated application of low concentrations of ryanodine triggered release at non-ribbon sites that varied in location from trial to trial. On the other hand, depolarization with KCl puffs, which stimulates release preferentially at ribbons , trigger2+ channels beneath the ribbon and stimulates the rapid release of vesicles  of vesicles tethered at the base of the ribbon is replenished when they remain hyperpolarized in bright light. Subsequent membrane depolarization accompanying a decrement in light triggers the opening of L-type Cavesicles . Releasechannels . During channels . After te ribbon .Figure 1) and by capacitance techniques . The evidence for substantial non-ribbon release from rods in retinal slices obtained by FALI experiments suggests that non-ribbon release observed by TIRFM in isolated rods is not an artifact of cell isolation procedures.chniques  indicatichniques  and the chniques  and the Consistent with a significant role for non-ribbon release from rods, electron tomography studies revealed frequent exocytotic omega figures in non-ribbon regions of rod synapses . In addi2+ stores has been implicated in driving sustained release from hair cells  which pumps Ca2+ from the cytoplasm (2+ entry also helps to regulate release from rods by elevating cytoplasmic Ca2+ and replenishing ER Ca2+ (Results from other polarized cells show that Cathe cell . FRAP excell (2+ . Consistytoplasm . Store-o ER Ca2+ . TogetheBy triggering release at non-ribbon sites, CICR increases the total amount of slow release from rods thereby amplifying the rate of release in darkness. Elevated release in darkness enhances decrements in release that occur when rods hyperpolarize to light. Use of distant non-ribbon release sites may also have a secondary benefit of reducing synaptic noise by diffusional filtering of glutamate to post-synaptic glutamate receptors . TogetheThe 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.


Additionally, the calcium release allows the N-lobe to rotate relative to the rest of the Tn molecule. Based on the findings presented herein we propose a novel model of skeletal thin filament regulation.The interaction between calcium and the regulatory site(s) of striated muscle regulatory protein troponin switches on and off muscle contraction. In skeletal troponin binding of calcium to sites I and II of the TnC subunit results in a set of structural changes in the troponin complex, displaces tropomyosin along the actin filament and allows myosin-actin interaction to produce mechanical force. In this study, we used molecular dynamics simulations to characterize the calcium dependent dynamics of the fast skeletal troponin molecule and its TnC subunit in the calcium saturated and depleted states. We focused on the N-lobe and on describing the atomic level events that take place subsequent to removal of the calcium ion from the regulatory sites I and II. A main structural event - a closure of the A/B helix hydrophobic pocket results from the integrated effect of the following conformational changes: the breakage of H-bond interactions between the backbone nitrogen atoms of the residues at positions 2, 9 and sidechain oxygen atoms of the residue at position 12 (N Troponin is composed of three subunits; troponin C (the Ca2+-binding subunit), troponin I (the inhibitory subunit) and troponin T (the tropomyosin-binding subunit) (2+ binding to the regulatory site(s) of TnC triggers a series of conformational changes in the troponin (Tn) complex, allows tropomyosin to move along the actin filaments and allows actin to interact with myosin to produce force The interaction between Casubunit) . Ca2+ bi2+-binding proteins with four Ca2+-binding sites. TnC consists of two globular lobes, the N- and the C-lobe, connected with a central linker. Each lobe contains a pair of EF-hand Ca2+-binding sites. The Ca2+-binding sites in the N-lobe are believed to be responsible for the regulation of muscle contraction, whereas those in the C-lobe show higher affinities for Ca2+ and play a structural role. In cardiac muscle, TnC has only one functional regulatory Ca2+-binding site (site II) in the N-lobe due to the replacement of the key Ca2+-coordinating amino acid residues in site I 2+-binding proteins with four Ca2+-binding sites and recognizes a number of target proteins, TnC is known to have only one Ca2+-dependent target protein - TnI. The switch region of TnI interacts with the N-lobe of TnC in a Ca2+-dependent manner.TnC is a member of the EF-hand Ca2+ directly or indirectly have been developed to treat patients with heart failure or with neuromuscular diseases 2+ binding to, or Ca2+release from TnC should help the effective development of such drugs.TnC has been a target for therapeutics. Drugs that affect TnC’s affinity for CaMolecular dynamics (MD) simulations are a suitable and effective approach to delineate key events during activation and relaxation at an atomic level. MD simulations explore the atomic level details of molecular motion of macromolecules by means of computing the Newtonian equations of motion for each atom to obtain the time dependent evolution of the molecular system. Such simulations, starting with the groundbreaking work on BPTI in the late 1970s EF-hand proteins have been the subject of several computational studies in recent years 2+ binding to Site I of cardiac Tn was observed to induce correlative motions in the EF-hand and domain movements that precipitate the forming of the TnI interface.Molecular dynamics has also been used in the study of the troponin complex. Recent works analyzed the correlated motions in the Tn subunits and Tn inter domain interactions 2+ at sites I and II in the N-lobe of TnC. Site I is a Ca2+-binding loop formed by TnC residues 29–40 and site II is a Ca2+-binding loop formed by TnC residues 65 to 76 . The key function of the EF-hand regulatory domain is to sense a Ca2+ chemical signal and undergo a conformational change which enables the further downstream transduction of the signal. In the case of TnC it has long been considered that this conformational change is a closure of the N-lobe A/B helix hydrophobic pocket and that in the absence of Ca2+ at the regulatory sites the N-lobe of TnC forms a “closed” structure; then upon Ca2+-binding to the regulatory sites, it adopts a “open” structure to provide TnI-interaction sites. Although these “final” structures have been investigated relatively well, the pathway between these two structures has not been understood. Additionally it has been proposed 2+ release from the TnC sites I and II, the TnI switch region is expelled from the TnC binding pocket which then closes. Upon Ca2+ binding, the pocket opens and the TnI switch region is re-inserted into the pocket. Despite the rich body of experimental and computational evidence unanswered questions regarding whether the EF-hand motif in general, and the TnC N-lobe in particular, is a bi-stable switch shuttling between open and closed states remain.Troponin binds Ca2+-dependent regulatory function of Tn 2+-depleted N-lobe with a fully closed hydrophobic pocket and in subsequent work 2+ causes the opening of the hydrophobic pocket for further interaction with TnI. In the case of cardiac TnC however the hydrophobic pocket of the N-lobe maintains a closed conformation after Ca2+ binds to site II while Site I does not bind Ca2+ at all, as was shown in several structural studies Indeed, blockage of the opening motion of the N-lobe by intra-domain disulphide bridging results in the practical disappearance of the Ca2+-dependent dynamics of the fast skeletal troponin molecule and the TnC subunit in the Ca2+-saturated and depleted states. We focused on the N-lobe and on describing the atomic level events that take place subsequent to removal of the Ca2+ ion from the regulatory sites I and II. This approach allowed us not only to compare the initial and final states of troponin but to also explore the dynamics of troponin in the activated and deactivated state; to explore the dynamics that take place subsequent to Ca2+ release; to reveal the atomic level details of the conformational changes that occur in response to Ca2+ release; and to characterize the transitional events leading to thin filament relaxation.In this study, we used MD simulations to characterize the Ca2+-saturated avian fast skeletal troponin molecule (1YTZ.pdb) 2+-depleted TnC subunit (5TnC.pdb) Molecular dynamic simulations were performed using the NAMD 2+- and apo-form crystal structures are available for the core domain of the skeletal troponin complex, 1YTZ.pdb is the only available structure in the Ca2+ form. The 5TnC.pdb crystal structure of Herzberg and James is the first crystal structure of EF-hand type Ca2+-binding proteins, is determined at 2 Å resolution, and is widely considered to be the gold standard for skeletal TnC structural description. The closeness of the two source species: Gallus gallus (1YTZ.pdb) and Meleagris gallopavo (5TnC.pdb) provided an additional level of complementarity. It should be noted that the selected PDB files have some missing residues. Specifically, in 1YTZ.pdb the 39 C-terminal amino acid residues (144–182) of TnI or the C-terminal residues of TnT2 (249–262), as well as the N-terminal tail domain of TnT, are not in the crystal set. We did not incorporate these residues in our simulations. These residues are considered to be flexible, at least in the absence of tropomyosin and it was demonstrated that the C-terminal mobile domain of TnI behaves independently from the core domain of the complex by NMR measurements Our choice of PDB system was driven by the considerations that while both Ca2+ present in sites I, II, III and IV (Tn4Ca2+); 2) core troponin , Ca2+ present in sites III and IV only (Tn2Ca2+); 3) TnC domain, Ca2+ present in sites I, II, III and IV (TnC4Ca2+); 4) TnC domain, Ca2+ present in sites III and IV (TnC2Ca2+); 5) TnC domain where site I was depleted, site II was saturated (TnCSite1noCa2+); 6) TnC domain, where site I was saturated, site II was depleted (TnCSite2noCa2+); 7) TnC domain, Ca2+ present in sites III and IV, with residue ALA24 mutated to ASP24 and LEU48 to GLU48 (TnCASP24GLU48), and 8) TnC domain, Ca2+ present in Site III and IV (5TnC). Protein initial coordinates for systems 1 through 7 were derived from 1YTZ.pdb and protein initial coordinates for system 8 were derived from 5TnC.pdb. Protein structure files (psf) were created using the molecular modeling package VMD In total we prepared and simulated 8 systems: 1) core troponin , Ca2+ release from the TnC N-lobe regulatory binding sites. A closure of the hydrophobic pocket results from the integrated effect of the following conformational events: breakage of H-bond interactions between the backbone nitrogen atoms of the residues at positions 2, 9 and the sidechain oxygen atoms of the residue at position 12 (N2-OE12/N9-OE12) in sites I and II; expansion of sites I and II and increased site II N-terminal end-segment flexibility; strengthening of the β-sheet scaffold and the subsequent hydrophobic re-packing of the N-lobe hydrophobic residues. Additionally the Ca2+ release allows the N-lobe to rotate 25–35 degrees relative to the rest of the Tn molecule.In this work we explored via molecular dynamics simulations the dynamics of the calcium sensor protein troponin and the events occurring after Ca2+ was removed from sites I and II, only from site I, only from site II and not removed at all (molecular coordinates derived from Ca2+-saturated structure - 1YTZ.pdb); second, the closed, deactivated TnC domain (molecular coordinates derived from the Ca2+-depleted TnC - 5TnC.pdb); and third, the troponin molecule Ca2+-saturated and Ca2+-depleted state (molecular coordinates derived from 1YTZ.pdb). Thus, we simulated the open activated skeletal Tn and TnC domain in the Ca2+-saturated state; and then to model the structural events leading to thin filament relaxation, we removed Ca2+ from sites I and II of open TnC and simulated the resulting structure. To access the conformational coupling between the two sites and the contribution of each site to the structural transitions of the N-lobe we also simulated structures with only one site occupied. The 5TnC based system served to establish a frame of reference to which to compare the structural transitions discerned from the simulations.We performed equilibrium molecular dynamics simulations on the TnC subunit (residues 3 to 161) and the core domain of the troponin complex. The following set of systems were examined: first, open TnC systems where Cad between the alpha-C atoms of the first residue of the A helix (GLU16) and the last residue of the B helix (LEU48). For the closed 5TnC system the pocket openness distance was d = 13.65 Å±0.71 (averaged over the duration of the simulation). For the open, Ca2+-depleted TnC system the pocket rapidly closed to d = 13.23 Å±0.60 (averaged over the last 10 ns of the 3 TnC2Ca2+ simulations). The open, Ca2+-saturated systems TnC pocket closed to d = 15.74 Å±1.29 (averaged over the last 10 ns of the 3 TnC4Ca2+ simulations). The open, site I-depleted, site II-saturated TnC system (TnCSite1noCa2+) closed to d = 16.48 Å±0.86 (averaged over the last 10 ns of the simulation), whereas the open site I-saturated, site II-depleted TnC system (TnCSite2noCa2+) closed to d = 15.18 Å±1.10 (averaged over the last 10 ns of the simulation). In the presence of TnI, the TnC pocket did not close in either Ca2+-saturated or depleted systems for the duration of the MD simulations and remained at d = 23.87 Å±0.89 and d = 23.79±0.94 Å respectively (averaged over the duration of the 3 Tn4Ca2+ and the 3 Tn2Ca2+ simulations).The level of openness of the TnC N-lobe hydrophobic pocket in the holo and apo state and the closing of the pocket can be observed and measured in a molecular dynamics simulation trajectory. To establish a consistent metric across all our simulations we measured the openness of the pocket as the distance 2+ and reached a closed state only if the regulatory sites were unoccupied  of TnC N-lobe residues (residues 5 to 80) were measured and are presented on 4Ca2+) in Ca2+-saturated state the observed RMSF at the saturated Ca2+-binding sites does not exceed 1Å , the depleted site shows significant increase of fluctuations vis-à-vis the saturated site. Additionally in the TnC simulations another RMSF peak was observed centered at residue GLN50. Thus the level of RMSF of each binding site depends on its Ca2+-state and the RMSF peak present in the B/C connected loop region is accounted by the movement of B/C helices towards the D helix which precipitates the closure of the A/B hydrophobic pocket observed in the TnC simulations., 7 and 12 of Ca2+-binding loop at the two sites th position bidentate ligand is a highly invariant GLU residue. Particularly important for the regulatory function of the troponin switch is a set of interactions that occur between the Ca2+ ion, sidechain OE1 and OE2 of GLU12 (residues GLU40 and GLU76) and the backbone N atoms of the residues at positions 2 and 9 (ALA30/GLU66 and SER37/ASP73) (denoted here as N2-OE12/N9-OE12). Thus in the Ca2+-bound state the sidechain oxygen atoms of the GLU12 are held in towards the Ca2+-binding pocket by a bi-dentate charge-charge interaction to the Ca2+ ion and by H-bonds towards the main chain nitrogen atoms of the two loop residues.The Ca2+ is present in high concentration.The importance of this interaction for the regulatory function of the EF-hand motif in calmodulin has been underlined by mutational studies where the GLU was replaced by GLN 2-OE12/N9-OE12 interaction is stable and is not broken during our simulation trajectories whenever the binding site is occupied by Ca2+. The CD atom of the GLU12 side chain in both site I and II maintain a distance to the N2 and N9 at under 3 Å in the simulation trajectories of Ca2+-saturated Tn and Ca2+-saturated TnC. In the TnCSite1noCa2+ and TnCSite2noCa2+ simulations this distance also remains under 3 Å at the Ca2+-occupied binding site. The distance between the GLU12 OE and the N2 is the lesser of the two distances in all cases. Ca2+ presence in the binding site is necessary to maintain the N2-OE12/N9-OE12 interaction and the motions of the tops of the site- entering and exiting helix are coupled by the ion present in the site.The N2+ release from both binding sites is followed by the immediate breakage and disbanding of the N2-OE12/N9-OE12 H-bond interactions . In contrast the TnCSite1noCa2+ simulation showed the similar N2-OE12/N9-OE12 breakage events at site I, however since site II is occupied by the ion, its ligands 1, 3 and 5 are closely held and the N-terminal segment of site II is less flexible. Hence we did not observe a closure of the pocket to the lowest d but only to an intermediate value. The opposite was observed in the TnCSite2noCa2+ simulation and since in this simulation the site II N-terminal end-segment was more flexible, the hydrophobic pocket was able to close tighter in our TnCSite2noCa2+ TnC simulation vis-à-vis the TnCSite1noCa2+ TnC simulation.At the same time of the N2-OE12/N9-OE12 interaction is followed by a dramatic hydrophobic rearrangement and repacking, which occurs at the A/B hydrophobic pocket. Residue PHE28’s side chain is previously hidden in the hydrophobic core of the protein and in contact with PHE77, LEU41 and VAL44. The increased flexibility in N-terminal end-segment of site I allows the PHE28 aromatic ring to rise up and out and emerge now on the outside of the pocket  the PHE28’s side chain and then inserts deep into the hydrophobic core of the N-lobe by making a contact with PHE77, PHE25  and ALA24. The side chain of the VAL44 finishes positioned under the aromatic ring of the PHE28 and into PHE77.3 ends of their side chains orient toward one another and seal the hydrophobic packing of the A/B pocket from below. THR43 rotates its side chain so that the hydroxyl group is towards the solvent and the methylene group is towards the inside of the pocket where it will meet the rising side chain of the PHE28.The two opposing methionines MET27 and MET47 change the relative position of their side chains and MET27’s side chain moves towards and slides under the side chain of MET47. The flexible S-CHAdditional conformational changes and hydrophobic rearrangement occurs resulting in a tighter hydrophobic packing between ILE60, ILE61, VAL64 on the C helix, ILE72 in the site II loop and PHE77, MET80 and MET81. MET80 side chain slides under ILE60 and packs towards LEU41 on the B helix. MET81 adjusts its flexible end to form a contact with LEU48 since the two residues end up close to each other as a result of the closing motion of the B/C helices. The three methionines MET81, MET45 and MET80 meet and the side chain of the MET45 inserts in the groove between the other two MET residues of the D helix, with the flexible end segments of all three residues sampling different rotamers until a tight packing fit is reached .In the holo state troponin complex and TnC simulations we observed steady hydrogen bonding between ILE36O-ILE72N and ILE36N-ILE72O while the donor-acceptor distance between GLY34-PHE74 and THR38-GLY70 remained well over 4 Å. Ca β-sheet . The THR2+-saturated or semi-saturated TnC reaches a semi-closed conformation in the absence of TnI. In this state the B/C helices move towards the A/D helices, the hydrophobic pocket opening is tightened and the opening distance reduced. The saturated TnC N-lobe however is not fully closed to the degree observed in the simulations of Ca2+-depleted state. The semi-closed state is a conformation resulting from the partial closure of the N-lobe within the constraints imposed by the Ca2+ ion present in the site I and II Ca2+-binding pockets , MET45(B) and MET80(D) are further separated in the semi-closed than in the closed state.Structural comparison of the open and semi-closed state reveals that the N2+ simulation where the release of the ion allows the formation of the THR38N-GLY70O H-bond.Additionally, the THR38N-GLY70O H-bond at the β sheet in the closed state is well formed at a distance of approximately 2 Å whereas in the semi-closed and open state, the distance is stable at over 4.7 Å, except in the case of the TnCSite2noCa2+. The existence of this state brings evidence to the innate ability of the TnC N-lobe to undergo a partial closure even when the trigger Ca2+ ion is present. The key difference however is that the removal of Ca2+ breaks the N2-OE12/N9-OE12 ‘prong’ interaction; liberates the N-terminal end segment of the binding site II thus allowing the B helix VAL44 to slide swiftly under the rising aromatic ring of the A helix’s PHE28 and make a contact with the PHE77 of the unperturbed hydrophobic core scaffold  of the N-lobe.In our simulations the TnC subunit collapsed to a semi-closed state accessible in the absence of TnI and the presence of Ca2+ modulated regulatory function of troponin. The closure motion would likely be very sensitive to residue mutations that would disrupt the hydrophobic fabric of the pocket. To test this notion as to prevent the closing of the TnC hydrophobic pocket when the Ca2+ ion is removed we mutated two hydrophobic residues into charged residues. Residue ALA24 and residue LEU48 were mutated into hydrophilic residues - ASP(24) and GLU(48)   A. The m GLU(48)  B. The s2+ from binding sites I and II in the TnC N-terminal domain induces a rotation of the TnC N-terminal domain relative to the rest of the TnC domain and to the IT arm of the troponin molecule. We measured the rotation of the TnC N-terminal segment relative to the C-terminal TnC segment and the rest of the Tn by comparing virtual dihedral angles formed by planes determined by alpha-C atoms suitably selected from different points of the Tn molecule  is presented for TnI to remain in close contact with the closed N-lobe pocket. Subsequent events may include melting of the TnC connecting helix as suggested by Vinogradova et al. 2+ ligands will reach 2-OE12/N9-OE12 interactions and inducing the B helix to move away from the A helix while unpacking the hydrophobic core. TnI switch region is then readily re-inserted in the opened pocket. Rotation in the opposite direction occurs at the same time. The combined opening-rotation repositions the TnI inhibitory region away from actin and subsequently exposes the actomyosin interaction sites.Based on our findings we propose a model for thin filament regulation. The deactivation of troponin can be interpreted as a closure-rotation conformational change occurring in response to CaThis study focused on the troponin molecule in isolation, as have other recent computational studies of troponin Figure S1Open, semi-closed and closed states of TnC N-lobe. A. Structural comparison of the open and semi-closed states of TnC N-lobe. (shown in red and orange respectively). B. Structural comparison of the semi-closed and closed state of TnC N-lobe . The helices of the N-lobe are labeled accordingly and the top panels show a ‘bird’s eye’ view of the N-lobe while the bottom panels show side views. The Ca2+ ions are shown as vdW shapes and in red and orange depending on if they are located in the open or semi-closed state structures. There are no Ca2+ ions in the closed state structures.(TIF)Click here for additional data file.Figure S2A mutation to prevent the TnC hydrophobic pocket from closure. A. Mutation of TnC N-lobe hydrophobic pocket residues ALA24 and LEU48 to ASP and GLU, respectively. The mutated residues are shown in surface representation and are seen protruding into the pocket. B. The evolution of the openness of the hydrophobic pocket of TnCASP24GLU48 in the Ca2+ depleted state as measured by the distance d between the alpha-C atoms of residues GLU16 and GLU48.(TIF)Click here for additional data file.Figure S3Rotation of the TnC N-lobe. A: Virtual dihedral 1 is determined by alpha-C atoms of residues GLU131, PHE104, ASP73, GLU15. The intersection of the two planes formed by alpha-C atoms of  and alpha-C atoms of  runs along the α-helix connecting the N-lobe and C-lobe of TnC. Residue 131 is located in the C-lobe of TnC. Residue 104 and 73 form a segment that spans the TnC connecting helix and Residue 15 is located at the start of the A helix. B: The virtual dihedral 2 is determined by alpha-C atoms of residues GLU131, PHE104, ASP73, GLU54. The intersection of the two planes formed by alpha-C atoms of  and alpha-C atoms of  runs along the α-helix connecting the N-lobe and C-lobe domains of TnC. Residue 131 is located in the C-terminal domain TnC. Residue 104 and 73 form a segment that spans the TnC connecting helix and Residue 54 is located at the start of the C helix. Alpha-C atoms of residues GLU131, PHE104, ASP73, GLU75, GLU15 and GLU54 of TnC shown as green vdW spheres. C. (Left panel) Time dependency of Virtual dihedral 1. Red line depicts the Tn4Ca2+ structure and blue line depicts theTn2Ca2+ structure. Tn2Ca2+ simulation was started from the equilibrated Tn4Ca2+ structure at 8 nanoseconds (marked with *). C: (Right panel) Time dependency of Virtual dihedral 2. Red line depicts the Tn4Ca2+ structure and blue line depicts the Tn2Ca2+ structure. Tn2Ca2+ simulation was started from the equilibrated Tn4Ca2+ structure at 8 nanoseconds (marked with *).(TIF)Click here for additional data file.Table S1List of systems, number and duration of simulations performed.(PDF)Click here for additional data file.Movie S1Hydrophobic core segment: PHE25, PHE74 and PHE77.(MPG)Click here for additional data file.Movie S2Expulsion of residue PHE28.(MPG)Click here for additional data file.Movie S3Movement of residues MET81, MET45 and MET80.(MPG)Click here for additional data file.


To assess this issue, a series of experiments was performed in vitro using the isolated spinal cord preparation from the neonatal rat with measurements of [Ca2+]o and extracellular potassium concentration ([K+]o) during pharmacologically induced fictive locomotion. We demonstrated that with the onset of fictive locomotion, [Ca2+]o reduced from 1.2 up to 0.9 mM whereas [K+]o increased from 4 up to 6 mM. At the same time, a special study performed on the isolated genetically identified Hb9 excitatory interneurons showed that, at [Ca2+]o= 1 mM and [K+]o=5 mM, 12% of Hb9 cells expressed intrinsic INaP-dependent bursting, and at the concentrations typical for fictive locomotion ([Ca2+]o= 0.9 mM and [K+]o=6 mM), as many as 50% of identified Hb9 interneurons expressed INaP-dependent bursting. Importantly, the threshold of [Ca2+]o to generate bursting decreased as [K+]o increased. The analysis of Hb9 neuron behavior during slow ramp increase of voltage revealed that lowering [Ca2+]o from 1.2 to 0.9 mM induced a negative shift (~ -3 mV) in the INaP half-activation voltage (V1/2NaP). In contrast, V1/2NaP was not changed when [K+]o increased from 4 to 6 mM.The spinal interneurons in newborn rodents, when synaptically isolated by removing the extracellular calcium (Ca+o), demo2+]o and [K+]o on the Hb9’s pacemaker properties and firing behavior, we developed a single-compartment computational model of Hb9 neuron. In this model, we explicitly simulated a negative shift of V1/2NaP occurring with the reduction of [Ca2+]o.. At [K+]o=6 mM, our model exhibited tonic activity at V1/2NaP = –50 mV . The rhythmic bursting emerged at V1/2NaP = –51 mV, and further shifting V1/2NaP to the left produced stable bursting . In turn, an increase in [K+]o reduced the potassium reversal potential and hence all voltage-gated potassium currents (IK), which provided an additional augmentation of INaP-dependent bursting [a synergistic effect of [Ca2+]o and [K+]oon the emergence of bursting activity, we modeled a population of 50 uncoupled neurons with randomly distributed parameters (see Fig. V1/2NaP towards more negative values induced by reducing [Ca2+]o may play a major role in emergence of bursting activity in the population of spinal interneurons. We have also demonstrated that accumulation of [K+]ocan facilitate the emergence of INaP-dependent bursting via the reduction of IK.To theoretically investigate the effect of changing o and [K+]o may convert activity of spinal interneurons from asynchronous/tonic to the synchronized bursting. This activity-dependent switching in firing behavior may represent a fundamental mechanism for locomotor rhythm generation in the spinal cord.In summary we suggest that co-regulation of


We describe the case of 18 year old twin brothers who presented to our unit with a 3 year history of aquagenic urticaria. This rare form of urticaria usually presents within an hour of contact with water. The aetiology is unknown. Most cases are sporadic but there are a small number of familial cases in the medical literature. A specific genetic mutation has not yet been found. To our knowledge, this is the first report of aquagenic urticaria in monozygotic twins, further supporting a genetic component to this disease. We describe the case of 18 year old twin brothers who presented with aquagenic urticaria. This is a rare form of urticaria which usually presents within an hour of contact with water. The aetiology is not understood. Most cases are sporadic but there are a small number of familial cases in the medical literature although no specific genetic mutation has been found yet. To our knowledge, this is the first report of aquagenic urticaria in monozygotic twins, further supporting a genetic component to this disease.18 year old identical twin brothers were referred to our tertiary referral centre urticaria clinic with a three year history of aquagenic urticaria. They were both keen triathletes and were therefore frequently exposed to water. Swimming or showering would induce a prodrome of skin pruritus followed by the development of wheals over the trunk approximately 15 minutes after the initial contact with water. The wheals were induced by both hot and cold water. They would resolve spontaneously after about one hour, leaving no residual mark. Neither brother had ever experienced any associated angioedema. Exercise such as running would only induce itching. There was no history of atopy and both brothers were in good general heath and were not taking any regular medications. The family history was unknown as the brothers had been adopted. On examination, the skin of both brothers was normal but we were able to induce the classical wheals following exposure to water in a shower in our clinic Figures . The symAquagenic urticaria is a rare form of urticaria which affects women more commonly than men. Symptoms often start in puberty. Wheals usually develop within 30 to 60 minutes of contact with water, irrespective of the temperature. Differential diagnoses include aquagenic pruritus, where exposure to water induces pruritus but no visible skin lesions, and cholinergic urticaria, where wheals develop in response to heat, exertion, sweating or emotional stress. The aetiology is not understood. It has been suggested that water interacting with sebum or sebaceous glands can produce a toxin causing degranulation of mast cells, leading to histamine release and wheal formation. An alternative explanation has been that water-soluble antigens in the epidermis diffuse into the dermis and cause a histamine mediated reaction. Oral antihistamines are used as first line treatment, up to four times the standard single daily dose. Successful treatment with UVB and PUVA has also been reported.Although almost all cases of aquagenic urticaria are sporadic, there are a small number of familial cases in the medical literature-7. Some Treudler er al reportedTo the best of our knowledge, this is the first case report of aquagenic urticaria in monozygotic twins, further supporting the notion that there is a genetic component to this type of urticaria, as suggested by others. It is hoped that new generation sequencing/exome sequencing will be able to shed further light on the genetic traits involved.Written informed consent was obtained from the patients for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in Chief of this journal.The authors declare that they have no competing interests.CF provided the case history. AK carried out the literature review and wrote the discussion. All authors read and approved the final manuscript.


Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca2+. Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca2+ elevation is not influenced by alkamides.With a traditional medical use for treatment of various ailments, herbal preparations of E. purpurea root extracts and constituents as potential regulators of intracellular Ca2+ levels. Changes in cytosolic Ca2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.A non-immune human cell line, HEK293, was chosen to evaluate 2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca2+ elevation is sustained after the Echinacea-induced Ca2+ release from intracellular Ca2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.A rapid transient increase in cytosolic CaEchinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.Our data indicate that as yet unidentified constituents from  Echinacea produces local anesthesia of the mucous membranes; thus, it has been used medicinally since ancient times i) were measured by ratiometric imaging techniques using a perfusion system as previously described i (Δ[Ca2+]iMax) was determined as the difference from the resting value to the maximum response. The time-to-peak value was measured and defined as the time it took for the signal to reach from the resting value to the maximum response. The response length was defined as the time interval from the time the signal reaches the maximum response to the time it took to return to the resting value. The dose-response curve was generated using PRISM 4.02 . The half-maximum concentration of Echinacea extract that induces a response (EC50) was calculated from the fitted sigmoidal curves using PRISM 4.02.Intracellular calcium concentrations ([Caescribed . Cells w2+] increase data (Δ[Ca2+]i) were represented as mean ± SEM, n = 3; data were compiled from cell traces from three biological replicates (three independent cell cultures), measuring at least 20 cell traces/replicate; each value is the mean of ≤ 20 cells from one replicate. Means of Δ[Ca2+]iMax were analyzed by t-test to determine statistical significance compared to the control. One-way analysis of variance followed by the Tukey test was also carried out to compare means between different treatments. All statistical analyses were performed using SAS software version 9.1 .The cytosolic i increase in HEK293 cells . Transient [Ca2+]i increases were also induced with similarly prepared extracts from E. pallida, E. angustifolia and E. tennesseensis (data not shown).s Figure . This Ect Figure , indicattization  Similarld Figure . To invensient Ca+i increa2+]i, E. purpurea extracts were fractionated using preparative HPLC. Twenty-eight fractions were collected and tested for bioactivity. Aliquots of the HPLC fractions were applied to HEK293 cells at levels proportional to their concentrations in the initial ethanol extract. In this HPLC protocol, phenolics such as cichoric acid and cholorogenic acid elute in the more polar fractions (retention times of about 2-40 min), whereas Bauer alkamides 1, 2, 3, 4, 8, 9, 10, 11 and Chen alkamide elute in the later less polar fractions (retention times of 49-94 min) [E. purpurea fractions  are active in evoking [Ca2+]i elevation in HEK293; the other 22 fractions have no detectable bioactivity  responsible for inducing this transient increase of cytosolic i increase in HEK293 cells by Echinacea. These responsible bioactive constituent(s) could be novel or alternately they might be identified in other plant species but not yet found in E. purpurea; for example, in E. pallida non-polar ketones such as pentadeca--dien-11-yn-2-one have been recently identified in E. pallida i increase depends on external calcium, HEK293 cells were perfused either with HEPES solution supplemented with normal concentrations of calcium (2 mM) or with EDTA-chelated calcium-free HEPES buffer for 10 min, before treatment with E. purpurea extracts. In both of these sets of experiments the Echinacea-induced transient increase in [Ca2+]i was observed  receptor, however, other mechanisms exist as well i. 2-APB (100 μM) completely abolishes the transient increase in [Ca2+]i evoked by the Echinacea-extract  activation is required for the t Figure , whereast Figure , consistEchinacea-extracts, external Ca2+ was removed and internal Ca2+ stores were depleted using the sarcoplasmic/ER Ca2+ -ATPase (SERCA) pump inhibitor, thapsigargin. Restoration of external Ca2+ following thapsigargin-treatment indicates the function of SOCE, which can be observed as an increase in cytosolic Ca2+ [Echinacea extract. The Ca2+ responses evoked by Echinacea and thapsigargin are similar  plays a role in calcium homeostasis in the HEK293 cell response to lic Ca2+ . Here, tr Figure . In the s Figure , 6B. Thiigure 2+ .Echinacea-induced Ca2+ entry pathways are pharmacologically similar in the HEK model, 2-APB was applied in the continued presence of external Ca2+. 2-APB is considered the most potent and consistent compound in blocking SOCE, acting as a potent SOCE inhibitor independent of, and in addition to, its ability to inhibit the IP3 receptor [2+ channels [2+ entry pathways [2+ entry [Echinacea extracts  was applied for 10 min and then the Echinacea extract (100 μg/ml) was applied to test whether there are any blocking effects of the antagonist. Our results showed that SR 144528 was not able to abolish the Echinacea response .This y Figure . Ca2+ inEchinacea might bind to a surface membrane receptor that is coupled to PLC, such as chemokine [E. purpurea extract have differential kinetic properties and potency on HEK293 cells  is consistent with more than one receptor being stimulated by Echinacea extracts. A second possibility is that bioactive constituent(s) enter the cell and act directly or via an intracellular moiety on PLC. These possibilities are not mutually exclusive. Because a portion of the Ca2+ response that is induced by Echinacea extracts is associated with lipophilic constituent(s), logical candidates for being permeable across cell membranes [A PLC-dependent signal might be mediated in several ways. For example, constituents of hemokine , glutamahemokine , or purihemokine . Microarhemokine . The obsembranes , we can 2+ channels in the ER membrane, usually via the IP3 receptor or ryanodine receptor families [2+ pumps located in the ER membranes then return cytosolic Ca2+ into the lumen, thus contributing to Ca2+ homeostasis between the cytoplasm and ER. The intracellular Ca2+ stores are refilled from the extracellular reservoir, mostly through SOCE [Calcium stored in the ER is released through Cafamilies . Ca2+ puugh SOCE . SOCE haugh SOCE .Echinacea-extracts is from internal stores, indicative of a metabotropic response. In this metabotropic model, PLC activation would lead to the production of IP3, which in turn would activate the IP3 receptor causing release of Ca2+ from the ER [3 receptor in the Echinacea-induced calcium release is suggested by treatment of HEK293 cells with the specific membrane-permeable IP3 receptor antagonist, 2-APB [Echinacea-induced calcium release from internal stores. 2-APB has been reported to enhance leakage of Ca2+ from the ER and inhibit SERCA activity, resulting in enhancement of Ca2+ signaling [Our data indicate that the source of the initial transient increase in cytosolic calcium levels that are induced by m the ER . The part, 2-APB . 2-APB iignaling . This coEchinacea-induced release of Ca2 from internal stores may be coupled to a subsequent activation of the SOCE process. In many cell types, depletion of intracellular Ca2+ stores results in the opening of SOCE in the plasma membrane [2+ [2+-store content to the opening of this Ca2+ channel, remains unclear. Two proteins have been implicated in SOCE function: Orai1, a pore-forming subunit of the SOCE, and stromal interaction molecule 1 (STIM1), thought to be an ER-based Ca2+ sensor that activates SOCE by an as yet undefined mechanism [2+ in ER after Echinacea treatment would in turn activate the plasma membrane SOCE through a mechanism that involves STIM1. Taken together, this model predicts that in HEK293 cells, the level of cytosolic Ca2+ associated with Echinacea treatment increases through two mechanisms: initially the release of Ca2+ from ER Ca2+ stores, and subsequently Ca2+ influx via SOCE.This model predicts that the membrane . SOCE, trane [2+ , is consrane [2+ . The natechanism . Therefo2+ increase, whether Echinacea-induced or otherwise, are complex and highly dependent on the cell type and context in which they occur. Longer range effects of changes in cytosolic levels of Ca2+ regulate a wide variety of cellular processes [2+ activate Ca2+-dependent enzymes, such as calcineurin, and thereby transcription factors, such as nuclear factor of activated T cells (NFAT) and nuclear factor-κB (NF-κB). These transcription factors modulate the activation of T-cells and generation of cytokines, which in turn regulate the expression of many target genes in inflammation and pain transmission [The physiological events downstream of a cytosolic Carocesses . In T-cesmission .Echinacea extracts enhance the cyclooxygenase 2 and cytokine signaling activities of various immune cells, both of which are involved in many steps of immunomodulatory responses and mediation of pain transmission [Echinacea preparations modulate the levels of varied cytokine transcripts in human acute monocytic leukemia cells, bronchial epithelial cells and dendritic cells [Echinacea preparations. Taken together, these studies and our own convey the important message that Echinacea may induce many responses in various cell types involving more than one signaling pathway, and that it is a combination of these responses that likely lead to the overall physiological effect on the organism.Studies using animal and human models indicate that smission ,11,19. Cic cells -31. The Echinacea components in a non-immune-related cell type that lacks CB2 receptors. Our use of a human non-immune cell line as an experimental system to evaluate Echinacea-induced response emphasizes the complex effects of herbal medicines, and sheds more light on potential molecular early signaling mechanisms for this important medicinal plant. Thus the non-CB-related intracellular calcium signaling induced by non-alkamide components of Echinacea extracts revealed in this study, in conjunction with the activation of CB-mediated signaling by Echinacea extracts by specific alkamides [This report highlights the effect of lipophilic, non-alkamide lkamides -8, proviEchinacea induce transient increases of cytosolic Ca2+ levels when applied to HEK293 cells, and that this increase can be attributed to a subset of lipohilic Echinacea constituents. HPLC fractionation reveals six distinct lipophilic fractions that induce this transient increase in cytosolic Ca2+. This bioactivity does not appear to be attributable to any known bioactive components of Echinacea, including the alkamides. Furthermore, it is not associated with the CB receptors. Based on studies with a range of inhibitors, activators and experimental conditions, we propose that Echinacea extract contains compounds other than its known alkamides that induce cytosolic Ca2+ release, in combination with an ER-depletion-associated activation of the SOCE pathway in HEK293 cells.In conclusion, we show that extracts from The authors declare that they have no competing interests.LW carried out the study design, experimental work on cell culture and calcium imaging data and conducted statistical analyses, literature search and drafted the manuscript. EWR participated in design and coordination of the study and provided assistance in data interpretation. KJ helped with the calcium imaging work. SJ provided assistance in data interpretation. LR did the GC-MS analysis of the plant extract and fractions. BJN evaluated the data and corrected the manuscript for publication. JM helped with the cell culture. MK provided assistance in data interpretation and edited the manuscript. ESW conceived the study, supervised the experimental work and co-wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/10/72/prepub


In contrast to the closely related Ca2+ indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca2+ and a sensing mechanism that does not involve direct modulation of the chromophore pKa. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca2+ binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the Kd for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.As part of an ongoing effort to develop genetically encoded calcium ion (Ca As the Ca2+ ion is a universal “second messenger”, detection of elevated Ca2+ levels can reveal when an intracellular signaling pathway is being activated . As. As23]. 3.2.3.Ka around pH 6.5 to 7.0. We hypothesized that, at physiological pH and in the absence of Ca2+, there is a specific residue (of unknown identity) for which the deprotonated form is quenching the fluorescence of the chromophore [2+ either this residue is no longer deprotonated due to a change in pKa, or it is no longer in the right conformation to quench the chromophore fluorescence. In an attempt to determine the identity of the residue that is so critical to the function of the CH-GECO variants, we performed mutagenesis of rationally selected candidate residues.From the CH-GECO2.1 pH titration curve reproduced in omophore . In the Ka in question is around 6.5 to 7.0, the most likely candidate residue was histidine. However, the X-ray crystal structure of mCherry [Kd for the variants that formed the red fluorescent chromophore. No red fluorescent protein could be obtained for Lys70Gln and Glu148Gln, and absorbance scans of both variants revealed that the chromophore maturation process was stuck at the blue intermediate species similar to CH-GECO2.1-PVV described above [2+ affinity that are similar to CH-GECO2.1. We conclude that this hydrogen bond network is not involved in the Ca2+-dependent response of the CH-GECO variants, but does play an important role in the chromophore maturation process.As the p mCherry  reveals  mCherry . We mutaed above . Mutatio2+ were determined  have reed mRuby . In the nd state , which iin RCaMP  is a metin RCaMP .2+ responsiveness, the Gln163Lys substitution was introduced into CH-GECO2.1-PVV-ATA. The resulting variant formed the red fluorescent chromophore but did not show a significant fluorescence response to Ca2+  in the 2+ free state, the side chain of Gln163 is directed outside the barrel similar to the orientation of Lys163 in R-GECO1 (2+. When CaM and M13 interact due to Ca2+ binding, the side chain of Gln163 moves inwards and interacts with the chromophore in a conformation similar to that observed in mCherry (2+-dependent switch from the exterior to the interior of the protein. In contrast, mutation of Gln163 to either Lys or Asp dramatically increases the energetic cost of bringing the charged side chain into the hydrophobic interior, and the Ca2+ sensing mechanism is abolished.We propose that, in the Ca R-GECO1 . In this mCherry . That is2+ of CH-GECO2.1 relative to R-GECO1, nor we were able to identify the key residue that is undergoing an ionization with a pKa ∼6.5 to 7.0. We suspect that, in both cases, the relevant residues are located in the interface between the CaM domain and the FP domain.Disappointingly, we were not able to unambiguously identify key residues the contributed to the much higher affinity for Ca4.2+ indicator CH-GECO2.1. While we were not able to answer all of the questions that naturally emerged from this effort, we were successful in identifying Gln163 as the most important residue for transducing the conformational change associated with Ca2+ binding into a change in the fluorescence intensity of the FP domain. We expect that this insight will be critical in guiding future efforts to further optimize genetically encoded Ca2+ indicators for improved response. Another important conclusion from this work is that interdomain connections and interactions can have a complex influence on multiple aspects of a genetically encoded biosensor. Specifically, we observed that changes in linker composition and length could effect protein maturation and the sensing mechanism. In addition, our results suggest that interfacial residues play a crucial role in determining the affinity of the indicator for Ca2+. We recommend that future efforts to optimize such indicators should always involve substantial screening of linker libraries that vary in both length and composition. Also, we suggest that, given the complexity of the sensing mechanisms, directed evolution efforts that involve screening of libraries of randomly generated variants will tend to be much more effective than efforts guided by naive mechanistic hypotheses.The goal of this work was to gain insight into the mechanistic details of the recently reported genetically encoded Ca


Furthermore, both non-selective protein kinase C (PKC) inhibitor and PKCα selective inhibitor, but not PKCδ inhibitor, prevented the increase in ERK1/2 phosphorylation and the migration of astrocytes, indicating that the Ca2+-dependent PKCα acts as the downstream of the OX1R activation and mediates the orexin-A-induced increase in ERK1/2 phosphorylation and cell migration. In conclusion, these results suggest that orexin-A can stimulate ERK1/2 phosphorylation and then facilitate the migration of astrocytes via PLC-PKCα signal pathway, providing new knowledge about the functions of the OX1R in astrocytes.Orexin-A is an important neuropeptide involved in the regulation of feeding, arousal, energy consuming, and reward seeking in the body. The effects of orexin-A have widely studied in neurons but not in astrocytes. Here, we report that OX1R and OX2R are expressed in cultured rat astrocytes. Orexin-A stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and then induced the migration of astrocytes via its receptor OX1R but not OX2R. Orexin-A-induced ERK1/2 phosphorylation and astrocytes migration are Ca However, they are now regarded as a syncytium of interconnected cells, rather than as individual bodies to maintain normal neurological functions. In most conditions, the effects of astrocytes are mainly mediated by their membrane receptors such as AMPA receptors Extracellular signal-regulated kinase 1/2 (ERK1/2) is a potential candidate for this supposition since ERK1/2 is reported to mediate orexin functions in several cell types 2+ contributed to orexin-A-induced ERK1/2 phosphorylation and astrocyte migration. In general, our results might provide new perspectives to understand the roles of orexin in central nervous system.In the present study, our results demonstrated the expression of OX1R and OX2R in the rat astrocytes. It was also shown that orexin-A promoted astrocytes migration by activation of ERK1/2 via augmenting OX1R-PLC-protein kinase Cα (PKCα) signals in cultured astrocytes. In addition, intracellular CaBAPTA-AM, U73122, 2-aminoethoxy-diphenylborate (2-APB), cyclopiazonic acid (CPA), GF109203X and poly-L-lysine were purchased from Sigma-Aldrich . Orexin-A, SB334867, Gö6976, Rottlerin and TCS OX2 29 were purchased from Tocris . U0126 was purchased from cell signaling . Fura-2/AM and Dulbecco's modified Eagle's medium DMEM/F12 were obtained from Biotium  and Gibco Invitrogen Corporation , respectively. Anti-ERK and anti-p-ERK antibodies were purchased from Cell Signaling ; Anti-β-actin, anti-OX1R and anti-OX2R antibodies were purchased from Santa Cruz . Anti-GFAP was purchased from Abcam . Both goat anti-mouse and goat anti-rabbit HRP IgG polyclonal secondary antibodies were purchased from Thermo Scientific . Other general agents were purchased from commercial suppliers.All the drugs were prepared as stock solutions. Orexin-A, TCS OX2 29 and poly-L-lysine were dissolved in the distilled water. Fura-2/AM, U73122, SB334867, 2-APB, U0126, GF109203X, Rottlerin, Gö6976, BAPTA-AM, and CPA were dissolved in dimethylsulfoxide (DMSO). All stock solutions were stored at −20°C. These stock solutions were diluted to the final concentrations with the extracellular solution before application. The final concentration of DMSO was <0.05%. No detectable effect of DMSO was found in the experiments.2 atmosphere incubator. Astrocytes were purified from the mixed culture by mild shaking , as described previously Neonatal Sprague-Dawley (SD) rats (day 0–3) were obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology. All animal care and experimental procedures complied with local and international guidelines on ethical use of animals and were approved by The University Animal Welfare Committee, Tongji Medical College, Huazhong University of Science & Technology. To obtain astrocyte, cortical tissues were isolated as previously described with some modifications After boiling in SDS sample buffer (5 min), equal amounts of protein were loaded in each lane and separated on 10% SDS/PAGE gels. Samples containing 20 µg proteins were separated electrophoretically and then transferred to nitrocellulose membranes by using a transfer cell system . After blocking with 5% nonfat dried milk powder/TBS/0.1% Tween 20 (1 h at room temperature), membranes were probed with the appropriate antibodies overnight at 4°C. Membrane-bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase (1∶10000). Immunoblots were developed on Microchemi  using the enhanced chemiluminescence technique . All assays were performed at least three times.2 1, CaCl2 2, Glucose 10 and HEPES 10 (pH 7.4) and incubated with 1.5 µg/ml Fura-2/AM for 30 min at 37°C. CaCl2 was omitted and 0.1 mM EGTA was added in Ca2+–free solutions. After additional washes, coverslips with Fura-2/AM-loaded cells were mounted on a chamber positioned on the movable stage of an inverted microscope  illuminated at 340 and 380 nm using calcium imaging system . Paired F340/F380 fluorescence ratio images were acquired every second for [Ca2+]i and the emitted light was imaged at 510 nm with a video camera (PTI Image) through a X-70 fluor oil immersion lens  and a 460 nm long-pass barrier filter. Ratio images (340 nm/380 nm) were analyzed by Metafluor software . The values were exported to Sigmaplot 10.0 for further analysis, and the [Ca2+]i response amplitude was normalized to the basal amplitudes, which was taken as 1.Primary astrocyte grown on glass cover slips were washed three times with extracellular solution containing (mM): NaCl 140, KCl 5, MgClAstrocyte cells were grown to confluence in dishes and starved with serum-free DMED/F12 medium for 24 h. The monolayer cells were manually scratched in the center of the dishes with a bright and clear field. The detached cells were removed by washing the cells once with PBS. Serum-free DMEM/F12 medium with or without orexin-A was added to each dish as indicated after pretreatment with the inhibitor for 30 min. Then the cells were separately fixed with 4% paraformaldehyde and labeled with anti-GFAP (1∶200) for photography and analyzing of the cell migration at 0, 24 and 48 h. Images of migratory cells from the scratched boundary were observed and acquired at different time with a digital camera and a light microscope . The images were exported to Sigmaplot 10.0 for further analysis, the boundary of cell migration was defined by average displacement from the dotted line, and the migration distance and migration area were normalized to the control level, which was taken as 1. The figure represents one of four individual experiments.Cultured astrocytes were fixed with 4% paraformaldehyde in 0.01 M phosphate-buffered saline  for 30 min and then rinsed three times with PBS for 10 min each. After that, they were then permeabilized in PBS with 0.3% Triton X-100 for 30 min, followed by 3% BSA-PBS for 30 min at room temperature. The cells were co-incubated with 1∶50 anti-OX1R, anti-OX2R, and anti-GFAP antibody in PBS/0.3% Triton X-100/1% BSA/2% goat serum overnight at 4°C. Cells were rinsed in PBS three times for 10 min each, and then co-incubated with different second antibody in PBS containing 0.3% TritonX-100, 2% goat serum and 1% BSA for 1 h at room temperature. After washed three times with PBS, cells were mounted on glass slides with 10%-15% glycerin and imaged using a confocal laser scanning microscope .5′-CAGATGAACTCTACCCTAA-3′; the negative control sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The astrocyte was infected with lentivirus at a multiplicity of infection (MOI) of 20 for 8 h. Then the medium was replaced with fresh complete medium. After 3 days, cells were observed under fluorescence microscopy to confirm that more than 80% of cells were GFP-positive.A third generation of Self-inactivating lentivirus vector  containing a CMV-driven GFP reporter and a U6 promoter upstream of the cloning sites (Age I and EcoR I) was used for cloning small hairpin RNAs (shRNAs). The target sequence for rat orexin receptor 1 (OX1R) was t test and ANOVA were used for statistical analysis where appropriate. The software SPSS 13.0 and Sigmaplot 10.0 were used. The criteria of significance were set at p<0.05.Data were expressed as mean ± SEM. Student's To investigate the effect of orexin-A on cell migration in astrocytes, the confluent monolayer of astrocytes was grown in serum deprived medium for 24 h, then dishes were scratched, added with different concentrations of orexin-A , then immunostained with anti-GFAP at 0, 24 and 48 h, respectively. It was found that orexin-A promoted astrocytes migration in different time and concentrations. It had a maximal effect at 10 nM and 24 h treatment  , suggestPrevious study have reported that orexin-A promotes cAMP synthesis in astrocytes via OX1R We then determined whether these receptors mediate orexin-A-induced increase in astrocytes migration. Firstly, OX1R siRNA was used to silence OX1R gene, the results showed that viral vector (null or carrying OX1R siRNA) had no effects on primary astrocytes migration, however, silencing OX1R gene significantly inhibited orexin-A induced cell migration . Next, wvs. 0.21±0.01 in control group, n = 3, p<0.05) in primary cortical astrocytes, suggesting the possible contribution of ERK1/2 pathway in orexin-A-induced migration of astrocytes. To test this hypothesis, ERK1/2 inhibitors U0126  and PD98059  were added before orexin-A incubation. The migration result showed that orexin-A-induced astrocytes migration was prevented by U0126 and PD98059 significantly  or vehicle for 10 min and the levels of ERK1/2 phosphorylation were assayed by western blot. As shown in ficantly .2+ participated in orexin-A-induced increase in ERK1/2 phosphorylation and subsequent cell migration in cultured astrocytes. Firstly, the changes in intracellular Ca2+ concentration by orexin-A were tested by calcium imaging. It was found that intracellular Ca2+ concentration increased notably about 500 sec after orexin-A (10 nM) application from 0.64±0.07 to 1.21±0.29 in fluorescence intensity  and store-operated calcium (SOC) channels 2+-ATPase) and 50 µM 2-APB (the inhibitor of InsP3Rs and SOC channel) 30 min before orexin-A administration respectively. The calcium imaging results showed that the orexin-A-induced Ca2+ elevation was inhibited significantly , followed by application of orexin-A (10 nM) for 10 min. As shown in vs. 0.86±0.05 in BAPTA-AM group, n = 4, p<0.05), it still prevented ERK1/2 phosphorylated continuously by orexin-A , suggesting that the increase of intracellular Ca2+ may contribute to orexin-A induced ERK1/2 phosphorylation. In order to confirm the role of intracellular Ca2+ in ERK1/2 activation, astrocytes were pretreated with different inhibitors. Firstly, Ca2+-free solution containing EGTA (the chelator of Ca2+) were administrated for 30 min before orexin-A treatment, we found that pretreatment of astrocytes with EGTA containing Ca2+-free solution significantly inhibited the increase in ERK1/2 phosphorylation in astrocytes , the inhibitor of endoplasmic reticulum Ca2+-ATPase, was also employed. Similar with BAPTA-AM, CPA also elevated the basal level of p-ERK , but differently, it could not inhibit the activating effect of orexin-A on ERK1/2, the p-ERK1/2 level still raised from 0.96±0.18 to 1.40±0.17   mediated these effects. It was shown that 2-APB , a SOCE inhibitor, prevented the increase of p-ERK expression by orexin-A from 0.59±0.08 (orexin-A group) to 0.22±0.07 (2-APB+orexin-A group)  , indicat2+ influx was required for orexin-A-induced astrocytes migration.Finally, pharmacological inhibitors of calcium origins BAPTA-AM, 2-APB were used to examine the effects of calcium signal on cell migration with wound healing assay. As shown in 2+, would affect orexin-A-induced ERK1/2 phosphorylation and migration. As expected, pre-incubation of 30 µM U73122, a specific inhibitor of PLC for 30 min, attenuated orexin-A -evoked ERK1/2 phosphorylation   . In line, n = 3) . These r2+-PKCα signaling pathway. Notably, astrocytes migration is important in brain inflammation and remodeling during brain injuries such as brain wound healing, tissue remodeling, and glial scar formation Orexin-A, a multifunctional neuropeptide in the nervous systems, is originally discovered in the hypothalamus in 1998 Astrocytes are involved in various pathological conditions through their membrane receptors such as the classical cannabinoid-like receptors Recent studies have reported that orexins can activate ERK1/2 associated signals, which plays crucial roles in the CNS 2+ level 2+ spike was appeared about 500 sec after orexin-A treatment in astrocytes. The results indicate that intracellular Ca2+ elevation may be involved in orexin-A-induced ERK1/2 phosphorylation and astrocyte migration. Such a long delay between adding orexin-A and the Ca2+ spike might due to indirect method of administration. However, when we used Ca2+ chelator BAPTA-AM to explore the detail mechanisms, we found that the basal level of ERK1/2 phosphorylation increased by BAPTA-AM, which was in accordance with our previous study 2+ increase and elimination of negative Ca2+-calcineurin signals by BAPTA-AM 2+ generally binds to calcineurin, which physically interacts with ERK1/2 and restrains its activity through de-phosphorylation 2+ signal participated in orexin-A-induced ERK1/2 activation. In addition, some calcium origin blockers had the similar inhibitory effects. Intracellular Ca2+ increase was mainly derived from two sources: the Ca2+ store mobilization 2+. After administration of EGTA (a chelator of extracellular Ca2+) containing Ca2+-free solution, orexin-A-induced increase in intracellular Ca2+ and ERK1/2 phosphorylation was considerably inhibited, suggesting that extracellular Ca2+ was necessary for orexin-A-induced ERK1/2 phosphorylation. The similar results were obtained by 2-APB (SOC inhibitor) administration. However, CPA (inhibitor of the endoplasmic reticulum Ca2+-ATPase) could not arrest orexin-A-induced astrocytes ERK1/2 phosphorylation. Taken together, these results showed that SOC-mediated extracellular Ca2+ influx was necessary for orexin-A-induced ERK1/2 phosphorylation and migration in astrocytes.In endothelial cells and CHO cells, OX1R activation causes a robust increase in intracellular Ca2+ elevation via both receptor-operated Ca2+ channels (ROCs) and PLC-Ca2+ release via SOCs 2+ from the ER and additional influx of extracellular Ca2+ through SOCs 2+ pathway via SOCs extracellular Ca2+ influx was needed for orexin-A-induced astrocytes migration.OXRs are known to induce intracellular Ca2+-dependent and activated by DAG, while, the novel PKCs are Ca2+-independent and also activated by DAG, but the atypical PKCs are both Ca2+ and DAG-independent PKC is an upstream kinase for ERK1/2 activation 2+-PKC pathway mediates the orexin-A-induced ERK1/2 activation and then contributes to migration in cultured rat astrocytes. It is demonstrated for the first time that orexin-A can regulate cell migration, which might help us to understand the new physiological and pathophysiological roles of orexin-A in the central nervous system.In conclusion, our results clearly demonstrated that OX1R-PLC-Ca


UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, has attracted a great deal of attention as a potent antitumour agent. Several clinical trials of UCN-01 alone or in combination with other agents for different tumour types are currently underway, and some of these trials have had positive results. Hepatocellular carcinoma has high incidence rates and is associated with poor prognosis and high mortality rates.Three different hepatoma cell lines  were treated with different concentrations of UCN-01, and the anti-tumour effects of UCN-01 were evaluated. Following UCN-01 treatment, cell growth was measured using an MTT assay, cell cycle arrest was assayed using flow cytometry, and the mechanisms of cell cycle arrest and invasion inhibition were investigated through western blotting and a Matrigel invasion assay.waf1 pathway, whereas the CHK2/CDC25C pathway and the p53/p21waf1 pathway were involved in the UCN-01-induced S phase arrest. UCN-01 remarkably inhibited Huh7 cell invasion in a time-dependent manner. Suppression of Huh7 cell invasion may be due to the down-regulation of phosphorylated β-catenin by UCN-01.After a 72-h UCN-01 treatment, the growth of different hepatoma cell lines was significantly inhibited in a dose-dependent manner, with IC50 values ranging from 69.76 to 222.74 nM. Flow cytometry results suggested that UCN-01 inhibits proliferation in the hepatoma cells by inducing S and G2/M phase arrest, but not G1/S arrest, which differs from previous reports that used other tumour cell lines. Western blot results illustrated that UCN-01 induces a G2/M phase arrest, regardless of the status of the p53/P21waf1 and CHK2/CDC25 pathways. Suppression of Huh7 cell invasion by UCN-01 may be due to the down-regulation of phosphorylated β-catenin. These data lend support for further studies on UCN-01 as a promising anti-HCC candidate.These findings suggest that UCN-01 induces hepatoma cell growth inhibition by regulating the p53/p21 Although substantial efforts have been made to improve the overall therapeutic outcomes for liver cancer, hepatocellular carcinoma (HCC) remains the fifth most common and the third most deadly cancer in the world. If HCC in vitro and in vivo. This antiproliferative activity may be through protein kinase C inhibition. Cell lysate protein content was determined using a Bicinchoninic acid (BCA) protein assay kit . The extracts (40 μg protein) were fractionated on polyacrylamide-SDS gels and transferred to PVDF membranes . The membranes were blocked with a solution containing 5% non-fat milk in PBS and incubated overnight with primary antibody at 4°C. Subsequently, the membranes were incubated with secondary antibody coupled to horseradish peroxidase . The reactive proteins were visualised using enhanced chemiluminescence  according to the manufacturer’s instructions. Western blot analyses were performed using standard procedures.t-test (P < 0.05).All values are expressed as the mean ± SD. The significance between mean values was evaluated using the two-tailed unpaired Student's 50 values, ranged from 69.76 to 222.74 nM  was evaluated using a cell viability assay  of UCN-01. However, total cellular p53 protein levels were not increased even up to 300 nM UCN-01 in any of the cell lines. FigureTo understand the mechanisms by which UCN-01 induces G2/M phase arrest, we studied intracellular signalling through western blot analyses Figure. UCN-01 Hepatoma cell invasion was assayed using the Matrigel invasion chamber. After 72 h, the lower layer membranes were fixed and stained, and the number of cells invading through the membrane was counted FigureA. Huh7 cIn this study, we investigated UCN-01 antitumour activity in three different hepatoma cell lines with a particular emphasis on the mechanism of the G2/M cell cycle arrest induction and invasion inhibition in Huh7 cells. There were several novel findings presented here. (a) Growth of all three hepatoma cell lines was significantly inhibited by UCN-01 (each with a different sensitivity), whereas there was no effect on the normal hepatic cell line. (b) UCN-01-induced S and G2/M phase (and not G1) cell cycle arrest altered the p53 and CHK2 pathways. (c) Increased phosphorylation of Chk2 Thr68 and p53 was critical for UCN-01-induced G2 arrest, while total CHK2 and p53 remained the same. (d) In Huh7 cells, which are p53 mutant and p21 defective, and in Hep3B cells, which are p53 defective, S phase and G2/M cell cycle arrest is induced by UCN-01, suggesting that the G2/M arrest is p53 independent. (e) In UCN-01-treated Huh 7 cells, invasion activity was significantly inhibited, which may be correlated with the down-regulation of phosphorylated β-catenin at Ser 552.Streptomyces as a selective protein kinase C inhibitor[UCN-01 was originally identified in nhibitor and was nhibitor-22. By inhibitor,23,24. Snhibitor-29.There have been reports that UCN-01 inhibits the growth of various human cancers, e.g., leukaemia, colon, and pancreatic cancers, through the induction of a G1 arrest;-33 howevHere, we focused on elucidating how UCN-01 induces G2/M arrest in Huh7, HepG2, and Hep3B cell lines. Among these cell lines, Huh7 is p53 mutant and p21 defective, and Hep3B is p53 defective, enabling us to determine whether the cell cycle arrest is p53 dependent.The cyclin B/Cdk2 complex is critical in regulating the cell cycle transition from G2 to M phase and is controlled by phosphorylation at various sites. In our waf1 protein levels in the HepG2 cell line. As a crucial cell cycle regulator, the p53 tumour suppressor has an important role in the cellular response to platinum agents[waf1 pathway is also involved in the G2/M cell cycle arrest of HepG2 cells.However, we found that UCN-01 induces p53 phosphorylation at Ser 15 and increased p21m agents,39. For m agents. P53 tram agents,42. Amonm agents-45. Our Interestingly, when UCN-01 was used to treat Huh7 cells (p53 mutant and p21 deficient) and Hep3B cells (p53 deficient), the G2/M arrest is still observed. These findings suggest that, although these two pathways both contribute to cell cycle arrest, the CHK2-Cdc25c pathway may have a more important role in these p53-deficient cell lines.In our study, UCN-01 induces G2/M cell cycle arrest through these two pathways in the Huh7, HepG2, and Hep3B cell lines Figure, but doeFinally, we found that Huh7 cell invasive activity was significantly inhibited by UCN-01. Recent studies indicate that phosphorylated β-catenin has a critical role in the invasion and development of many different tumours-49. β-cain vivo, UCN-01 has decent activity against several tumours at dose levels that are tolerated in clinical studies[in vitro and in vivo and may be clinically applicable for HCC treatment in spite of its binding to serum proteins.In summary, our results show that UCN-01 inhibited HCC cell growth through the induction of S and G2/M phase arrest. UCN-01-induced G2/M cell cycle arrest involved molecular alterations of cell cycle regulatory proteins in human hepatoma cells. Increased Chk2 phosphorylation at Thr68 was critical for UCN-01-induced G2 arrest. These findings indicate that UCN-01 modulates the G2 transition through the p53-p21waf1 pathway as well as the Chk2–Cdc25c–Cdc2/cyclin B1 pathway. We also found that cell invasion was significantly inhibited by UCN-01 in Huh7 cells, which may be related to the down-regulation of phosphorylated β-catenin. Overall, the current study demonstrates that UCN-01 may be a potential drug candidate for the treatment of hepatocellular carcinomas.Although recent studies indicate that UCN-01 binds to the human serum protein alpha1-acid glycoprotein and that this binding may hamper the ability of UCN-01 to inhibit cell proliferation and kill cells  studies,28. ThesThe authors declare that they have no competing interest.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/167/prepub