Proteins, with their kinetic models, are located within two segments of dendrites containing 88 intact spines, centered in a fully reconstructed 6 × 6 × 5 μm3 cube of hippocampal neuropil. Protein components include AMPA- and NMDA-type glutamate receptors, L- and R-type voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, plasma membrane Ca2+ ATPases, smooth endoplasmic reticulum Ca2+ ATPases, immobile Ca2+ buffers, and calbindin. Kinetic models for each protein were taken from published studies of the isolated proteins in vitro. For simulation of electrical stimuli, the time course of voltage changes in the dendritic spine was generated with the desired stimulus in the program NEURON. Voltage-dependent parameters were then continuously re-adjusted during simulations in MCell to reproduce the effects of the stimulus. Nine parameters of the model were optimized within realistic experimental limits by a process that compared results of simulations to published data. We find that simulations in the optimized model reproduce the timing and amplitude of Ca2+ transients measured experimentally in intact neurons. Thus, we demonstrate that the characteristics of individual isolated proteins determined in vitro can accurately reproduce the dynamics of experimentally measured Ca2+ transients in spines. The model will provide a test bed for exploring the roles of additional proteins that regulate Ca2+ influx into spines and for studying the behavior of protein targets in the spine that are regulated by Ca2+ influx.We have built a stochastic model in the program MCell that simulates Ca Both MCell and CellBlender are available at mcell.org.The model was encoded in version 3.2 of the MCell Monte Carlo simulator  were defined by first tracing the contours of each PSD and active zone (AZ) on each original micrograph on which they appeared. The stack of such contours for a given PSD-AZ pair encloses the synaptic junction. The reconstructed contours include the intracellular extent of both the PSD and the AZ and intersect with the pre- and postsynaptic membrane associated with each. Therefore, we used the outer boundaries of the contours as a “3D-lasso” to tag surface mesh triangles representing the postsynaptic membrane and the location of the associated PSD , and closed at the place where the neck of the spine meets the dendritic shaft. Numbers of molecules within each counting box, and the flux between each spine and the shaft were measured at each time step of the simulations (1 μs), except for Ca2+, which was measured every 0.1 μs. All counts were recorded in an output file every 100 μs. Counts of molecules were sometimes converted to concentrations by converting to moles and dividing by the volume of the spine after subtracting the volume of any ER or mitochondria that intersected the sampling volume.MCell permits counting and tracking of molecules within a defined volume and also tracking of molecular fluxes across a defined surface. In the simulations reported here we tracked molecules of free CaNa, IK, IKM (K channel blocked by Ach acting through the muscarinic receptor), IKCa , PMCA pump mediating Ca2+ decay. Its geometry consists of the soma, 11 primary dendrites, and 87 branches, divided into 164 compartments. Details are available in the NEURON ModelDB .The simulations reported here were initiated by release of glutamate from a single site on the presynaptic membrane  were simulated by injecting current into the axon hillock and recording the voltages experienced at a spine located on a dendritic branch ~100 μm from the soma, as described in Keller et al. . The NEUProteins were placed onto the membrane surfaces of the reconstructed dendrites or in the volume of the cytosol as shown in the cartoon in Figure 2+ influx through NMDARs and VDCCs. AMPARs are included in the simulation because AMPARs compete with NMDARs for binding of glutamate. Thus, the competition for binding of glutamate is modeled directly, allowing an accurate estimate of the activation of NMDARs. The density of AMPARs was set to 1200 μm−2 of PSD surface (120 receptors for a PSD of diameter 350 nm). In vivo, the number of AMPA receptors in spines is highly variable and is altered by synaptic plasticity . This number is consistent with estimates of receptor numbers per PSD in the literature  curve indicating the Ca2+ current through the open channel at each voltage. For the calculation, Ca2+ conductance was set at 4.5 pS (10% of 45 pS) and ENMDA was set to 3 mV  or by bAPs. For the stimuli used in the simulations reported here, they are the largest source of Ca2+ influx. The precise complement of VDCCs in spines and shafts of pyramidal neurons is uncertain for two reasons. First, subtypes of VDCCs are sometimes difficult to separate pharmacologically. For example, CaV1.3 channels (one of two L-type channels) are difficult to distinguish from CaV2.3 channels (R-type) because both are resistant to inhibition by dihydropyridines  was calculated from Equation (2)  has the units s−1.The rate constant −2 in apical dendrites when measured in membrane patches , we first calculated the volume (V) from the molecular masses of Fluo4 and OGB1  and their densities (~1.4 g/cc). Both values were obtained from LifeTechnologies.com. We then assumed that We calibrated parameters of the model by fitting the characteristic of simulated Ca2+ indicators and pharmacological agents indicate that Ca2+ is removed from the spine cytosol by the combined action of three transmembrane proteins, Na+/Ca2+ exchangers (NCX's), smooth endoplasmic reticulum Ca2+ ATPases (SERCA's), and plasma membrane Ca2+ ATPases (PMCA's).Experiments with fluorescent Ca2+ (KD ~ 3 μM), but a large exchange capacity with turnover numbers sometimes as high as several thousand per second  at which a pump with Ca2+ bound extrudes Ca2+ from the cell, was setat 600 s−1. The concentration of Ca2+ at which the pump operates at half maximum velocity (KM) was set at 3 μM  and use the energy of ATP hydrolysis to reduce the Ca2+ concentration to its resting level with a turnover rate of ~12 s−1 measured in vitro  activates VDCCs in a 5 μm length of dendrite, we assume that the electrical length constant of the dendrite would cause the bAP to activate VDCCs in the neighboring length of dendrite equally, causing Ca2+ ions to flow into and out of the segments at an equal rate.Accurate modeling of a biochemical reaction–diffusion system requires appropriate treatment of the boundary conditions. We used a Neumann boundary condition; that is, we made the truncated ends of the two simulated dendritic segments reflective to Ca2+ concentration was maintained at 100 nM by adjusting the leak rates of the PMCA's and NCX's  at 100 nM Ca2+ that is related to the fraction of pumps with bound Ca2+ (fb) at that concentration: 2+, both at rest and after a stimulus, depends on the Ca2+ concentration. The quasi-steady-state assumption , or 100 μM OGB1 to dendrites, as appropriate . To accurately simulate the data of Sabatini et al.  on the smaller diameter dendrite (seven spines) for calibration and in all spines on the larger diameter dendrite for later tests. The individual simulations were averaged for each spine, and then the means from each spine were averaged and the standard error of the mean was determined. This procedure closely recapitulates the statistical analysis of the experimental data (see Section Results). The random placement of membrane molecules for each simulation with a new random seed introduces more variability into simulations in each spine than would be observed in repeated scans of the same spine in an experiment, because it introduces stochastic variation of channel numbers within each individual simulated spine. Thus, the simulations mimic sampling of 100 spines with the same shape and size, but slightly different numbers and arrangements of proteins.One hundred individual simulations, each with a new random seed, were carried out with each set of parameters. Ca3 (0.03 fl) and the average base-tip length of spines was 0.85 μm. These measurements are consistent with those of Harris and Stevens , at each concentration of Ca2+ indicator. The endogenous buffering capacity, κe, of spines and shafts was then calculated by the method of Neher and Augustine  for transients in the spine head, and the same two values for transients in the shaft , xi and yi are the ith observation of output values x or y, respectively. Likewise, μx and μy are the mean output value for x or y, across n = seven spines.We next refined the local fit and determined approximate parameter sensitivities using a form of Principal Component Analysis as follows. First, we constructed three 4 × 4 covariance matrices  for the three sets of four output values determined from the simulation data we collected from the seven spines. The calculations are summarized in Equations (10–12), where whereand2+ transients by fluorescence methods.We then averaged the three matrices together, element by element, to create an average covariance matrix. For this calculation, it would be ideal to use the experimental covariances. However, because we don't have access to the raw experimental data, we instead computed the average of the three covariance matrices from the simulation results. To the extent that we have chosen the correct arrangement of proteins for the simulations, this matrix should be similar to an experimental covariance matrix. The averaging reduces the dimensionality of the aggregate “output value space” from 12D (3 × 4D), which would be underdetermined by our seven variable parameters, to 4D, which is, instead, overdetermined by the seven variable parameters. This method is adequate for estimating parameter sensitivies and checking the goodness of fit, given the boundaries on the variable parameters, and the inherent noisiness of the original experimental measurements of Caz, the Z-score in four dimensions). To do this calculation, the origins of the four eigenvectors with lengths scaled to 1 sigma, as derived from the corresponding eigenvalues, were placed at the four-dimensional point representing the four target experimental values. Then the distances (z) between the four experimental target values and the corresponding output values for the 14 sets of parameters was calculated by projecting the 14 four-dimensional points onto the four scaled eigenvectors. The ratio of the length of each projection to the length of the eigenvectors is a measure that represents the number of standard deviations, or Z-score, of one of the simulated values from the target value  in the spine head or shaft). This operation projects, or transforms, the four original values into a new four-dimensional measurement space where the four transformed values are the coordinates of a point, m, whose distance, z  from the point representing the target value is given by the equation:i, is the ith dimension of point m, the superscript s indicates the transformed simulated value, and superscript t indicates the transformed target experimental value. The change in z, Δz, over the fractional change in parameter value p, p = 1.5p − 0.9p or 0.6 × the preliminary best fit value. In the language of Principal Component Analysis, this calculation means that the difference vector between simulated and target measurements was projected onto the four principal components, and the resulting difference vector components were normalized by the standard deviations of these principal components.We know from computational statistics and Principle Component Analysis that the four eigenvalues of this covariance matrix are the interdependent variances of each of the four output measurements in the direction of the eigenvectors (axes of the principle components). Thus, the square root of the eigenvalues equals 1 standard deviation  in the direction of its associated eigenvector. We used the eigenvalues and eigenvectors to refine the fit and determine the parameter sensitivities by the following method. We ran 14 simulations in which each of the seven parameters was varied independently either increasing it to 1.5 times its preliminary best fit value, or decreasing it to 0.9 times its preliminary best fit value (14 groups of parameters). These increments were chosen so that the resulting change in the output value was large enough not to be occluded by the Monte–Carlo trial-to-trial variability, but small enough to ensure rough linearity between the change in output value and the change in the parameter value. These simulations were run for each of the three indicator concentrations. For each set of parameters, the four output values were calculated for each of the seven spines, averaging 100 simulation runs. Then the mean output values were averaged over the seven spines to obtain three sets of four averaged output values for each set of parameters at the three indicator concentrations. The three sets of four values were then averaged to obtain a single set of four values for each of the 14 sets of parameters. The target  values were represented as a point in four-space and each group of output values for the 14 sets of parameters was also represented as a point in four-space. The radial distances from the point in four-space representing the target  values to the point in four-space for each set of parameters was then calculated, and normalized by the square root of the corresponding eigenvalue   and extrapolated to minimum z. The output values calculated from the simulations showed a small improvement in fit and are plotted in Figures To further improve the fit, we re-adjusted each parameter in the initial best-fit parameter set by an appropriate amount to decrease the final distance e) in the same range as reported by Sabatini et al.  than in the small shaft, and the decay time is longer, reflecting the smaller surface to volume ratio of the shaft with the larger diameter.After we calibrated the model to match experimental data using the small dendrite, we used the same parameters to simulate Ca Figures . The mos2+ indicators, they can be removed from the simulations to explore Ca2+ dynamics in the absence of indicators. We know that exogenous indicators alter the free Ca2+ transient, but their effect can't be measured by direct experiments. We used the calibrated model to simulate and compare free Ca2+ transients in a small , in response to three different stimuli; an EPSP alone, a bAP alone, and an EPSP preceding a bAP by 10 ms  and absence (green) of calbindin , the peak amplitude was nearly 13 μM and the time to decay to 10% of the peak was 50 ms.After a simulated EPSP+bAP stimulus Figure , only zee Figure . In the 2+ influx at spine #37 after an EPSP+bAP stimulus in the absence of calbindin is shown in Figure 2+ flows in through an opened NMDA receptor . Only a tiny fraction of Ca2+ remained free in the spine (blue). Ca2+ that bound to pumps was eventually pumped out of the spine (red and white) unless it unbound before being pumped. Ca2+ bound to immobile CBPs was released gradually to be captured by pumps or to diffuse out of the spine neck either while free or while bound to calbindin (green).During the first 100 ms of a bAP stimulus, we monitored the fate of all the Ca Figures . For com Figures . The Ca22+ exits the median-size spine than the large spine by diffusion into the shaft. The principal effect of calbindin is to decrease the fraction of Ca2+ exiting via the pumps and to accelerate diffusion of Ca2+ into the shaft while it is bound to calbindin. Sabatini et al. , the time required for complete equilibration between the spine and shaft is likely to be longer than 1 s. Because the Ca2+ transient decays much faster than Ca2+ is cleared by diffusion, they concluded that diffusion through the spine neck is not likely to be a major determinant of the decay time of a Ca2+ transient produced by a bAP. Our simulations of Ca2+ movements are consistent with this conclusion.As expected, a larger fraction of total Cai et al.  reportedin vitro, of the major proteins believed to control the influx and efflux of Ca2+ in spines can account for the measured size and decay time of Ca2+ transients in a computer reconstruction of intact dendrites and spines. We have shown that the properties of these proteins can indeed account for the size and decay of the Ca2+ transients when incorporated into our model. This conclusion comes with several qualifications. Some of the required parameters, in particular, the densities of individual classes of proteins in subcellular membranes, are difficult to measure experimentally. Hence, we have sometimes used molecular numbers, and/or membrane protein densities measured in non-neuronal cells, to establish biologically reasonable densities. For example, the densities of Na+/Ca2+ exchangers (NCXs) and plasma membrane Ca2+ ATPases (PMCAs) on the membrane of the spine head have not been measured directly. In our calibrations, we found that the required density of PMCAs in the spine head is higher by a factor of 2–3 than their measured density in plasma membranes of other cell types (see below). The values of the densities of NCXs and PMCAs that fit our calibration depend, in part, on their assigned turnover numbers. However, it is not possible to be certain that the enzymatic turnover numbers measured in vitro for these two pumps accurately reflect the turnover numbers in vivo. For example, if the turnover rate for PMCAs is higher in vivo than the rate measured in vitro, a lower density of PMCAs would be sufficient to account for the transients observed in vivo. Nonetheless, all of the final parameters in this model are within reasonable physiological limits. Thus, we conclude that the set of proteins we have used in the model, to a first approximation, can serve as the principal regulators of Ca2+ transients in spines. In addition, we conclude that the properties of these proteins, measured in vitro in the studies we have chosen (see Methods Section), are not likely to be grossly different than their properties displayed in vivo. Another area of experimental uncertainty is the immobile endogenous buffer, which represents the aggregate concentration and binding rates of the set of cytosolic proteins that bind Ca2+. The concentration (79.7 μM) and KD of the CBP that we established during the calibration are reasonable considering the high total protein concentration in the cytosol, and the weak binding properties of most immobile Ca2+ binding proteins. Because the concentration of the immobile buffer is not well-constrained by experiments, one might wonder whether a different concentration of CBP's would result in an equally good fit with a different set of values for the other parameters. However, there was not another good fit within the range of each of the parameters that we searched : 1. PMCAs are present at a higher density in spines than in non-neuronal cells; 2. they have a higher turnover number in vivo than measured in vitro; or 3. there are additional factors important for removing Ca2+ from the spine that are not well-understood by molecular neurobiologists. We favor the idea that PMCAs are expressed at unusually high levels in the spine membrane because spines have evolved to exert tight control over Ca2+ fluxes.The distribution of PMCA and NCX pumps between spine and shaft is qualitatively consistent with studies showing that Ca2+ transients under particular conditions, or in particular synaptic types. These components include SK channels, calcium-permeable AMPARs, metabotropic receptors, and inositol trisphosphate (IP3) and ryanodine receptors in SER. SK channels hyperpolarize the spine membrane in response to an initial Ca2+ influx, and thus strongly modulate the influx of Ca2+ during repetitive stimulation . We have not modeled those spines in the present study. It will be interesting to examine the effect of the presence of Ca2+-permeable AMPARs and Ca2+ stores on spine Ca2+ transients in future studies.The model presented here doesn't include several additional spine membrane components that can modulate the timing and/or amplitude of Ca2+-handling permits accurate simulation of diffusion of Ca2+ and mobile Ca2+-binding proteins through spine necks and into the nearby dendritic shafts, assuming a fairly uniform cytosol. However, we have not included agents that might mediate changes in the diffusional barrier in the spine neck, such as highly cross-linked actin cytoskeleton or mobile organelles that might move transiently into the neck  was best fit by two exponential curves, one fast and one slow. As the on-rate of the immobile CBP was increased and approached the on-rate of the indicator, the shape of the decay phase of the fluorescence transient was better fit by a single exponential. Furthermore, the off-rate  of the immobile CBP also had to be balanced against that of the indicator to avoid a biphasic exponential decay of the fluorescence transient. This phenomenon can be understood by noting that during the rising phase of a transient, the buffer or dye with the higher on-rate will initially bind most of the Ca2+, but during the decay phase the buffer or dye with the lower KD  will eventually bind the most Ca2+  the estimation method assumes that the data acquisition and subsequent lowpass filtering of the fluorescence signal are fast enough to track the fluorescence transient. The model shows that both of these assumptions are false. Ca2+ does not approach equilibrium with the indicator during the transient, and the Ca2+ peak is significantly attenuated by the lowpass filter.To understand and model the biochemical responses to Ca Figures . The err2+ fluxes into a median size spine and a large spine  reveal that, although a larger amount of Ca2+ flows into the larger spine, the effective concentration of free Ca2+ reached in that spine is smaller because the Ca2+ is averaged over a much larger volume. Our future studies will examine in more detail, in different sizes and shapes of spines, the relationship between the surface to volume ratio of a spine and the concentrations and local interactions of Ca2+ in the spine cytosol after a stimulus.The comparison of Ca Figures , 9 revea2+ handling in spines provides a test bed in which the sensitivity of Ca2+ transients to changes in the numbers and parameters of other modulating proteins can be predicted both independently and in combination. The model will also enable future studies in which calmodulin and its target enzymes will be added to the spine cytosol to test and predict how Ca2+ influx, triggered by a wide variety of stimuli, can affect activation of Ca2+-regulated enzymes that control synaptic plasticity.The model presented here will be a useful adjunct to experimental studies of the regulatory systems that control synaptic strength. For example, the basic model of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The molecular identity of the mitochondrial permeability transition pore (MPTP), a key player in cell death, remains controversial. Here we use a novel MPTP inhibitor to demonstrate that formation of the pore involves native mitochondrial membrane proteins adopting novel conformations. Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca2+], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca2+. However, when GNX-4975 was added after Ca2+ it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca2+ binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its ‘c’ conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT  when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca2+]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore.Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant ( These include elevated [Pi], adenine nucleotide depletion, agents such as carboxyatractyloside (CAT) that stabilize the adenine nucleotide translocase (ANT) in its cytoplasmic facing (‘c’) conformation, mild chaotropic agents such as KSCN and oxidative stress or its chemical mimic phenylarsine oxide (PAO) . Indeed, CyP-D is the site of action of two potent inhibitors of MPTP opening, cyclosporin A (CsA) and sanglifehrin A (SfA), both of which protect cells from death mediated by MPTP opening such as in ischaemia/reperfusion injury   . We propUnless otherwise stated, all chemical and biochemicals were obtained from Sigma–Aldrich or Fisher Scientific. Antibodies against the C-terminus of the ANT and PiC were raised either in-house or commercially as described in . The use2+] sensitivity of the MPTP caused by progressive loss of total adenine nucleotides over the first few hours of storage on ice  to open the MPTP, the extent of which was then determined by the rate of shrinkage induced by addition of polyethylene glycol (PEG)   and A520 was monitored continuously in a spectrophotometer with computerized data acquisition. When GNX-4975 was included, it was added 2 min prior to the addition of Ca2+, whereas incubations with other drugs or reagents were carried out for 4 min prior to the addition of Ca2+.This was performed by one of two techniques. A swelling assay was used in which pore opening was triggered by addition of Ca2+ ,28. In aol (PEG) . In both2. Any residual swelling was terminated by an addition of 1.2 mM EGTA, which also resealed swollen mitochondria. The resulting swollen mitochondria were collected by centrifugation at 12 000 g for 10 min and resuspended at 2 mg/ml in de-energized KSCN buffer without added NTA or A23187. In order to ensure equilibrium of matrix with the buffer, the swollen mitochondria were incubated again at 30°C supplemented with 1 mM CaCl2. After 3 min, 1.2 mM EGTA was added to reseal the mitochondria before centrifugation at 12 000 g for 10 min. The swollen mitochondria were then resuspended at 30 mg/ml in de-energized KSCN buffer containing 2 mM NTA and 2 μM A23187 and stored on ice. The extent of MPTP opening in these pre-swollen mitochondria was determined following incubation at 0.33 or 0.67 mg/ml for the stated time in KSCN or KCl buffer containing, where indicated, the required [GNX-4975] and free [Ca2+]  were pre-swollen by incubation for 20 min at 30°C in standard de-energized KSCN buffer (as above) but without added NTA or A23187 and with addition of 1 mM CaClA520 change. As described previously  with Fura-FF fluorescence, membrane potential with Rhodamine 123 fluorescence and mitochondrial swelling with light scattering at 490 nm as described previously  (Fura-FF fluorescence), membrane potential (Rhodamine 123 fluorescence) and mitochondrial swelling (light scattering at 490 nm) as described previously  within the mitochondrial matrix is a key trigger for MPTP opening and, under de-energized conditions, the extent of MPTP opening can be increased progressively with increasing [Ca2+] [2+] used to trigger MPTP opening on the number of GNX-4975-binding sites. When MPTP opening was triggered with 150 μM [Ca2+], initial rates of swelling were 200±7.9% of the rates at 50 μM [Ca2+] and the corresponding number of GNX-4975-binding sites increased from 13.2±2.4 to 20.9±1.6 pmol/mg  without any significant change in Ki value (1.91±0.35 and 2.15±0.26 nM respectively). These data imply that [Ca2+] may trigger MPTP opening by inducing the open conformation to which GNX-4975 binds.Increased [Cag [Ca2+] ,32. In F2+. This places limitations on studying the interaction between [Ca2+] and GNX-4975 binding since the inhibitor must always be added before or at the same time as Ca2+. This limitation can be overcome by using a different assay of MPTP opening in which mitochondria are pre-swollen with Ca2+ and then caused to shrink by addition of PEG 2000 that is too large to permeate the MPTP. The PEG exerts an osmotic pressure on the IMM that shrinks the mitochondria at a rate (measured as an increase in A520) proportional to the extent of MPTP opening [2+ prior to addition of PEG 2000 which allows GNX-4975 to be added either before or after Ca2+ prior to determination of the extent of MPTP opening. We first demonstrated that the shrinkage assay could be used to determine the number and Ki of GNX-4975-binding sites on the active MPTP. Data are presented in Ki of 2.08±0.30 nmol (mean ± S.E.M. for three separate experiments). The number of GNX-4975-binding sites is substantially higher than that observed in the swelling data of 2+] in KSCN buffer which is accompanied by the loss of intramitochondrial ATP and ADP, both of which inhibit MPTP opening /Ki)) with the derived Ki values at 1 and 2 mg of total protein being 117±15 and 166±29 nM respectively .Having validated the shrinkage assay for measurement of GNX-4975 binding to the MPTP, we employed it to see whether the order of addition of CaKi values under all the conditions used in the present study. Reassuringly, differences in the calculated Ki values are small. We confirmed that these differences in the calculated Ki values could not account for any differences in calculated Et values by re-analysing the data for each experimental condition using a fixed value of Ki . These data are also included in r) of 0.743; P<0.001). This strongly supports the hypothesis that GNX-4975 binds to the open pore complex and that a variety of agents that enhance or reduce MPTP opening do so by increasing or decreasing this active conformation of the MPTP. By contrast, titration of MPTP opening with CsA always produces the same number of binding sites irrespective of the degree of activation because it inhibits by interacting with CyP-D in its native conformation, preventing its binding to the membrane component(s) of the MPTP  increase the number of inhibitor-binding sites, whereas the BKA and ADP that reduce MPTP opening decrease the number of GNX-4975-binding sites. None of these effectors caused a major change in the Ki of GNX-4975 for inhibition of MPTP opening provided the inhibitor was added before Ca2+ as in the swelling assay for MPTP opening. However, using the shrinkage assay, it was shown that if Ca2+ was added prior to GNX-4975 the Ki was greatly elevated  has also been observed for CsA binding [koff could be determined from the Ki if the rate constant for inhibitor binding (kon) was known, since Ki=koff/kon. Values of kon for tight inhibitor binding to soluble enzymes have been shown to range between 104 and 109 mol−1·l−1·s−1 and can be substantially lower where inhibitor binding produces a conformation change [kon of 105 mol−1·l−1·s−1 the calculated koff is 2×10−4 s−1 implying that it would take nearly 1 h for 50% dissociation of the inhibitor and thus that Ca2+ would not displace the GNX-4975 during the time of the assay. This would explain the low Ki values observed when the inhibitor was added prior to Ca2+, but considerably higher values when the order of addition was reversed.Our data demonstrate that GNX-4975 is a potent MPTP inhibitor  which share a common structure in which six transmembrane helices form the substrate translocation pathway that involves conformational changes between inward- and outward-facing conformations . Thus its and Pi .


This is due to the fact that the molecular mechanisms of “docking” and “priming” reactions, which set up secretory vesicles to fuse at millisecond time scale, are extremely hard to study. Yet, driven by a fruitful combination of in vitro and in vivo analyses, our mechanistic understanding of Ca2+-triggered vesicle fusion has certainly advanced in the past few years. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. In particular, we will focus on the role of the small regulatory factor complexin whose function in Ca2+-dependent exocytosis has been controversially discussed for more than a decade. Special emphasis will also be laid on the functional relationship of complexin and synaptotagmin, as both proteins possibly act as allies and/or antagonists to govern SNARE-mediated exocytosis.Despite intensive research, it is still unclear how an immediate and profound acceleration of exocytosis is triggered by appropriate Ca The core membrane fusion machinery is constituted by a set of three highly conserved proteins known as the SNAREs  attachment protein receptors) . The  outside , 5. The  outside –9. Compl outside , Ca2+-dereaction . Indeed,reaction . At actiiggering , detaileiggering , 15. HowCaenorhabditis elegans or Drosophila melanogaster express only a smaller number of complexin isoforms, which are sequence-wise closely related to the cplxI/II subfamily but frequently also contain a C-terminal extension with a CAAX farnesylation motif like cplxIII/IV i, should lead to a depletion of the vesicle pools due to unfettered exocytosis. Indeed, ultrastructural and high-resolution imaging studies have shown that loss of complexin results in a specific loss of membrane proximal vesicles at gans NMJ  and mousgans NMJ . Likewisgans NMJ , 59. Congans NMJ , a phenogans NMJ . In vitrgans NMJ , 63. Thagans NMJ . In cont the NMJ , 45. GivIn conclusion, despite some remaining uncertainties, the combined set of data from in vitro and in vivo studies provides a model where complexin takes center stage in clamping of premature vesicle release.cis-SNARE complexes i i . These oin [Ca]i , 78, 80,in [Ca]i , 78. Theomplexin , 81–83.Thus, phenotypic cues from the vast majority of model systems as well as in vitro analyses indicate a fusion-promoting action of complexin that either complements concurrent complexin-mediated “clamping” of spontaneous fusion or even represents its chief function depending on the particular model system.−/− hippocampal neurons , this s2+-sensitivity of evoked release has indeed been reported for complexin-deficient neurons/endocrine cells in most preparations i. This implies a push–pull mechanism, wherein a sytI/Ca2+-mediated acceleration of fusion pore dilation counteracts a cplxII ‘clamp’ action that counters pore expansion. Intriguingly, the C-terminal domain of complexin that is involved in suppressing premature fusion was found to be also responsible for clamping the fusion pore. Thus, sytI in its Ca2+-bound form overcomes cplxII-mediated restraints on force transduction at the moment of the Ca2+-rise to initiate formation and accelerate dilation of fusion pore—putting both proteins in a truly antagonistic relationship during the last steps of exocytosis. On the molecular level, the functional antagonism between complexin and sytI may be due to the induction of limited conformational changes upon Ca2+-binding, as both factors likely reside on the SNARE complex.If Caut cells . These rut cells , 118. Thdynamics . Althoug64A, D65A, D68A) responsible for binding the complexin central helix also interferes with sytI-induced fusion in an in vitro fusion assay. While the authors’ interpretation that throwing the ‘switch’ lifts a fusion clamp on a neighboring SNARE complex is highly controversial (see above), changing the relative position of the accessory helix and N-terminus might still be essential for switching the mode of complexin action. Given that the very N-terminus is fulfilling a facilitating function, such conformational changes might place the domain close to the C-terminal end of the SNARE complex where it promotes full assembly of the SNARE proteins or regulates the binding configuration of sytI. In both scenarios, it might exert a fusion-promoting function wherein sytI and complexin act as allies affecting vesicle fusogenicity and triggering. A related view on complexin function was recently offered by Erwin Neher, who suggested that it may act as an allosteric adaptor for sytI [2+-dependency of release. Following this line of argumentation, complexin loss may reduce the dynamic range of the Ca2+-dependent secretion by invoking changes in the energy levels of the Ca2+-bound (increased energy barrier) and its free states (decreased energy barrier).Though little is currently known about the conformational states involved in triggering, existing cues might at least provide welcome primers for future research avenues: Krishnakumar et al.  recentlyfor sytI . Importa2+:sytI, possibly activating the arrested fusion intermediate without dislodging complexin (Fig. In summary, current evidence points to clearly antagonistic roles of complexin and sytI in clamping, with Ca2+-triggered vesicle fusion, the underlying molecular mechanisms are still far from understood. Evidently, complexin is small but capable, because it seems to affect crucial players in the exocytotic machinery with its few domains. Nevertheless, based on our current knowledge, it needs to be stressed that the seemingly counter-intuitive combination of fusion-inhibiting and fusion-promoting functions within one small accessory factor has a tremendous synergistic impact on the fidelity of Ca2+-triggered secretion.Almost 20 years after its identification as an SNARE-interacting protein, complexin still remains an enigma. Even though it has become clear by now that complexin serves a dual function in vesicle fusion, namely clamping of premature release and facilitation of Ca


Dysregulation of this Ca2+ handling system as well as increases in the production of reactive oxygen species (ROS) are two major contributing factors to myocardial disease progression. ROS, generated by cellular oxidases and by-products of cellular metabolism, are highly reactive oxygen derivatives that function as key secondary messengers within the heart and contribute to normal homeostatic function. However, excessive production of ROS, as in disease, can directly interact with kinases critical for Ca2+ regulation. This post-translational oxidative modification therefore links changes in the redox status of the myocardium to phospho-regulated pathways essential for its function. This review aims to describe the oxidative regulation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase A (PKA), and the subsequent impact this has on Ca2+ handling within the myocardium. Elucidating the impact of alterations in intracellular ROS production on Ca2+ dynamics through oxidative modification of key ROS sensing kinases, may provide novel therapeutic targets for preventing myocardial disease progression.Homeostatic cardiac function is maintained by a complex network of interdependent signaling pathways which become compromised during disease progression. Excitation-contraction-coupling, the translation of an electrical signal to a contractile response is critically dependent on a tightly controlled sequence of events culminating in a rise in intracellular Ca These forms of ROS are capable of altering cellular signaling by modifying susceptible proteins. Initially thought to contribute solely to cellular damage it has become clear that ROS signaling pathways are in fact a highly organized and compartmentalized network critical for homeostatic biological function . Importantly, as mitochondrial complexes are abundant in heme groups and iron sulfur clusters H2O2 can also be converted to highly reactive hydroxyl radicals  and thus have a low pKa. The pKa of a cysteine thiol is determined by its local tertiary environment, which is lowered by close proximity to basic amino acids lysine, arginine, or histidine.The ability of ROS to act as secondary messengers is attributed to their ability to oxidize susceptible cysteine thiols or sulfur on methionine residues of select proteins. The alteration of cysteine or methionine residues by oxidation generates a conjugated moiety with a different shape and charge characteristic, which can induce structure rearrangement to alter enzymatic activity. Protein oxidation is considered a physiological signaling modality as it can specifically and transiently regulate protein function. The selectivity of protein oxidation is provided by the limited reactivity of cysteine thiols. This is due to the majority of cysteines being buried within proteins and thus not accessible to an oxidant. In addition the pKa of an accessible cysteine thiol is an important determinant of its reactivity. For a cysteine thiol to be sensitive to oxidation it generally needs to be in a deprotonated “reactive” thiolate anion form  intermediate, which is then rapidly resolved by an adjacent cysteine on the same protein or neighboring protein to form an intra- or inter-molecular disulfide respectively   Kim, .2H) and then to sulfonic acid (SO3H). The formation of a sulfonic acid is irreversible, whereas a sulfinic acid is only reversible on peroxiredoxin at the expense of ATP and driven by the enzyme sulfiredoxin  also contribute to thiol-dependent redox signaling by forming protein nitric oxide (NO) adducts termed S-nitrosylation (SNO). NO generated from L-arginine by the nitric oxide synthases while remaining in its native form is unable to directly modify cysteine thiols. However, the formation of the nitrosonium cation (NO+) or dinitrogen trioxide (N2O3) can induce direct thiol oxidation (N2O3 + RSH → RSNO + HNO2), whereas, small thiol containing compounds such as S-nitrosoglutathione (GSNO) can induce protein S-nitrosylation through an exchange reaction termed trans-nitrosylation (GSNO + RSH → RSNO + GSH) . Peroxynitrite can induce S-nitrosylation but also irreversible tyrosine nitration that is often associated with disease.In addition to HThe oxidative modifications induced by ROS and RNS account for a large proportion of cellular thiol-dependent redox signaling. However, this form of signaling is further extended by the ability of cysteines to be modified by reactive lipids and natural electrophiles. These modifications are predominantly dependent on the presence of an alpha-beta unsaturated carbonyl group that allows nucleophilic addition of a thiol through a Michael addition reaction. Examples of thiol reactive lipids include 15-deoxy-Δ12,14-Prostaglandin J2, nitro-oleic acid and 4-hydroxynonenal  and allowing Ca2+ to flow into the cell where it binds to cardiac ryanodine receptors (RyR2) on the adjacent sarcoplasmic reticulum (SR). This binding precipitates cell-wide Ca2+ release from the sarcoplasmic reticulum (SR) store termed “Ca2+ induced Ca2+ release” (CICR), giving rise to a synchronous cardiac contraction. For the myocardium to relax it is necessary that cytoplasmic Ca2+ levels are returned to diastolic levels allowing Ca2+ to dissociate from the myofilaments. This is accomplished through inactivation of extracellular Ca2+ entering through LTCC and Ca2+ extrusion from the cytoplasm via the sarcoplasmic reticulum ATPase (SERCA), Na/Ca2+ exchanger, sarcolemmal Ca2+ ATPase and mitochondrial uniporter . An action potential depolarizes the cell membrane activating voltage sensitive L-type Caer Bers, .2+ handling and thus ECC within cardiomyocytes. Following the development of cardiac or vascular pathology the contractile requirements of the heart are severely increased in order to adequately compensate for the diseased tissue. The abundance of LTCC is reduced and the channel becomes hyperphosphorylated  would be beneficial to the failing myocardium as it would compensate for low levels of systolic Ca2+. However, as the NCX also extrudes Ca2+ in exchange for Na+ its increased activity will introduce a depolarizing current to the cytosol, a mechanism contributing to the formation of arrhythmic delayed-after-depolarizations  to the regulatory domain, which occurs under high intracellular Ca2+ concentrations. This induces a conformational shift in the structure of the kinase which disrupts the association between the regulatory and catalytic domains allowing the catalytic subunit to phosphorylate its substrates.Each monomer of the dodecameric CaMKII multimer associates via its C-terminal domain to form a double ringed hexameric complex  even after the addition of a Ca2+ chelator  deficient mice. MyD88−∕− myocytes showed no increase in CaMKII oxidation in response to lipopolysaccharide and were protected against CaMKII oxidation, cardiac hypertrophy, inflammation, apoptosis, and mortality following myocardial infarction , thus providing a mechanism for compartmentalization and specificity of PKA signaling   can oxidize RIα resulting in vasodilation independent of cAMP or NO signaling pathways. Therefore, the activation of PKA by CysNO was anticipated to exert positive inotropic effects on the myocardium. However, this was not observed due to CysNO's concurrent oxidative activation of PKG1α which exerts a partial negative inotropic effect on the myocardium (Burgoyne and Eaton, The intracellular ROS environment is maintained through alterations in oxidant producing enzymes, peroxidases, and reducing proteins. Fluctuations in the redox status of biological systems directly and indirectly alter protein structure and function. Key mediators of cardiac physiological function that are subject to oxidative-modification are the kinases PKA and CaMKII. Oxidation of both kinases leads to their activation therefore translating intercellular oxidative fluctuations into phosphorylation-mediated signaling. Under conditions of excessive oxidant production these signaling pathways can become aberrant and may contribute to heart failure progression. Failure to thoroughly understand the precise chemical pathways underlying these changes may be responsible for the disappointing results of long-term anti-oxidant clinical trials. Full elucidation of the mechanisms involving protein oxidation will allow for the development of more specific therapies that may inhibit and/or cure cardiac disease progression.The 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.


Compared with control group, AngII could greatly stimulate the increase of intracellular Ca2+ level, the activities and protein expression of cardiomyocytes CaN, and the expression of proto - oncogene c - fos mRNA in cultured neonatal rat cardiomyocytes, which could be effectively decreased by buckwheat rutin. Our results demonstrated that buckwheat rutin exhibited inhibitory effect on AngII - induced hypertrophy in cultured neonatal rat cardiomyocytes via Ca2+ antagonism action thus block the CaN - dependent signal pathway.Buckwheat rutin has been found to be able to inhibit angiotensin II (AngII) - induced hypertrophy in cultured neonatal rat cardiomyocytes, but the mechanism remains uncertain. In this study, myocardial hypertrophy model was made by adding AngII to the medium of cardiac myocytes of neonatal rats; meanwhile, different concentrations of buckwheat rutin were applied to observe their effects. Intracellular Ca Myocardial hypertrophy, which often leads to congestive heart failure, severe arrhythmia and disruption of important artery branches, is a commonly seen complication of cardiovascular system diseases. It is an independent risk factor causing the increase of death rate of cardiovascular diseases, and it gravely threatens the physical and mental health and life quality of human being. etc. In recent years, the experts have thought highly of the imbalance of neurohumor factors such as catecholamine, endothelin-1, various growth factors, especially the renin-angiotensin-aldosterone system that causes myocardial hypertrophy. Generous investigations have manifested that AngII has growth factor like effect. It can cause the increase in total protein content of cardiac myocytes and in protein synthesis rate, which results in the hypertrophy of cardiac myocytes. It can also increase the synthesis and excretion of matrixes of myocardium interstitial cells (mainly cardiac fibroblasts) and causes myocardial hypertrophy or myocardial fibrosis. The elevation of intracellular Ca2+ level is indicated to be the central link of hemodynamics overload and various neurohumor factors inducing myocardial hypertrophy. Ca2+ - calmodulin (CaM) dependent CaN pathway is a recently discovered important signal transduction pathway, which directly participates in several extracellular signal pathways that cause myocardial hypertrophy.Myocardial hypertrophy is commonly caused by hypertension, vascular disease of the heart, coronary heart disease, myocardial infarction and congenital heart disease 3-GATA4 thus inhibit myocardial hypertrophy and improve congestive heart failure. Due to the impact of side effects, CsA has not been spreadly applied in the clinic.As to the treatment of myocardial hypertrophy, immunodepressant Cyclosporin A (CsA) is studied the most. CsA could effectively suppress the expression of myocardial CaN-NFATFagopyrum esculentum Moench.  with abundant resources and mature techniques. Our previous studies have found rutin could inhibit AngII - induced hypertrophy in cultured neonatal rat cardiomyocytes. To investigate the mechanism, intracellular Ca2+ level, the activities and protein expression of CaN, and the expression of proto - oncogene c - fos mRNA in cultured neonatal rat cardiomyocytes induced by AngII were determined. The finding could provide experimental evidence for the development of new drugs for prevention and cure of myocardial hypertrophy.Rutin, also named as globulariacitrin and rutoside, possess many pharmacological activities including lowering the capillary permeability, anti-inflammatory, anti-anaphylaxis, anti-tumor, anti-bacteria, anti-virus and hepato-protection. Buckwheat rutin is an active ingredient extracted from Plant materials and isolation of rutinBuckwheat, flowers and leaves collected in late autumn from Ku Lun, Inner Mongolia (China), was identified by the Department of Natural Medicinal Chemistry, Hebei United University, Tangshan, China. A voucher specimen is deposited in the Department of Pharmacology, Hebei United University, Tangshan, China. Buckwheat rutin was isolated with the method demonstrated in the literature .Cell culture and induction of hypertrophy2 humidified incubator. Hypertrophy of cardiac myocytes was induced by adding 10-7 mol/L AngII, a known promoting growth factor, to serum-free medium  supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% COExperimental groups(1)The primarily cultured cardiac myocytes were synchronized with serum-free DMEM for 24 h as they grew to the proper density, then the cells were grouped as follows:Control group: Cultured in serum-free DMEM.-7 mol/L Ang II.Model group: Cultured in serum-free DMEM supplemented with 10Rutin I group: Cultured in serum-free DMEM supplemented with 0.8 mg/L rutin 30 min prior to Ang II.Rutin II group: Cultured in serum-free DMEM supplemented with 4.0 mg/L rutin 30 min prior to Ang II.Rutin III group: Cultured in serum-free DMEM supplemented with 8.0 mg/L rutin 30 min prior to Ang II.Different dosages of rutin were chosen according to previous studies.Detection of intracellular Ca2+ level (2)2+ levels were detected with Fura-2/AM, the specific indicator of Ca2+. Briefly, cardiac myocytes were loaded with 4 µmol/L Fura-2 / AM (dissolved in dimethyl sulfoxide with 0.02% pluronic) for 30 minutes at 37 °C in a humidified incubator with 95% air /5% CO2. Cells were then washed three times with modified Hanks' buffer containing (mmol/L) NaCl 137, NaHCO3 4.2, NaHPO4 3, KCl 5.4, KH2PO4 0.4, CaCl2 1.3, MgCl2 0.5, MgSO4 0.8, glucose 10, and HEPES 5 (pH 7.4). Fluorescence was determined with the Hitachi - 850 fluorospectrophotometer using dual excitatory wavelengths of 343 and 380 nm and a single-emission wavelength of 520 nm. [Ca2+]i was determined with the equation of Grynkiewicz et al: [Ca2+]i=Kdxß(R-Rmin)/(Rmax-R), where Kd is the dissociation constant for fura 2 - Ca2+ and taken to be 224 nmol/L, ß is the ratio of fluorescence at 380 nm and zero Ca2+ (F380min) and saturating Ca2+ (F380max) conditions, and R is the ratio of fluorescence obtained with excitation at 343 and 380 nm, with min and max subscripts denoting the ratios obtained under Ca2+-free and Ca2+-saturated conditions, respectively. The unit of [Ca2+]i is nmol/ L. The test was repeated for six times. Cardiac myocytes were treated with various concentrations of rutin for 24 h, then, intracellular CaPreparation of cardiac myocytes and measurement of protein content Cardiac myocytes were dealed with drugs and AngII for 24 h, and then they were washed thoroughly with PBS for three times and collected, precooled homogenate  was added and the cells were broken into pieces by repeated freezing and thawing, and centrifuged at 4 °C, 12000 g for 10 min. The supernatant was obtained and protein content was measured by Brad-ford method. Measurement of CaN activity3 and 0.4 mmol/L EGTA were added to stop the reaction and the absorbance was measured by automatic microplate reader at 410 nm (results expressed as A410nm). The substrates were used for zero setting. Substrate I contained 50 mmol/L Tris-HCl PH 7.4, 0.5 mmol/L DTT, 0.2 mg/mL BSA, 10 mmol/L PNPP, 0.5mmol/L MnCl2, 0.2 mmol/L CaCl2, 0.3 μmol/L CaM. Substrate II contained 3 mmol/L EGTA without CaCl2 and CaM. A410nm with substrate I reflected the activity of total phosphatases, and A410nm with substrate II reflected the activity of other phosphatases except CaN. So, A410nm of CaN was their subtraction. CaN activity was expressed as A410nm/mgpr.CaN activity was measured by colorimetric method referred to the literature  with somCaN protein expression observed by immunocytochemistry5 cells/mL, 1 mL per hole) and cultured for 48 h prior to 24 h’ synchronization, dealed with drugs for another 24 h before termination, fixed with 4% paraformaldehyde. CaN protein expression was detected using mouse monoclonal antibody raised against rat CaN-α  as first antibody and biotin-labeled goat anti-mouse IgG as second antibody. Photos were analyzed by MoticMed System 6.0A .Primarily cultured cardiac myocytes were seeded in 24-well plate with 10 mm×8 mm sterile coverslips . Statistical significances were analyzed using the ANOVA test. A value of 2+]i in control group, model group, Rutin I group, Rutin II group and Rutin III group were 121 ± 22 nmol/ L, 178 ± 25 nmol/ L (P < 0.01 v.s. control group ), 153 ± 27 nmol/ L, 132 ± 21 nmol/ L (P < 0.05 v.s. model group ), 128 ± 24 nmol/ L (P < 0.05 v.s. model group ) respectively. Buckwheat rutin inhibited increase of [Ca2+]i in Ang II - induced cardiac myocytes remarkably.Results showed [Ca-7 mol/L Ang II -induced cardiac myocytes was twice more than that of control group. Buckwheat rutin at 0.8 mg/L, 4.0 mg/L, 8.0 mg/L could obviously inhibit the increasing of CaN activity caused by Ang II  and showed dose-dependent relationship , whereas, buckwheat rutin at dosages of 0.8 mg/L, 4.0 mg/L, 8.0 mg/L could greatly decrease the Ang II induced expression of CaN protein to 1.503 ± 0.064, 1.488 ± 0.071 and 0.822 ± 0.051 respectively (P<0.01) in a dose-effect relationship . After that, the expression of c-fos mRNA began to decrease gradually and nearly went back to normal level in 2 hours. This demonstrated that c-fos mRNA expression was stimulated by Ang II and existed specific time difference , while different concentrations of buckwheat rutin could obviously reverse these changes. Compared with Ang II group, c-fos mRNA expression in different concentrations of buckwheat rutin decreased 26% (P < 0.05), 36% (P < 0.01) and 47% (P < 0.01) individually  and inositol (3). DAG can activate PKC, as a result, the cell membrane ion channel Ca-L opens and Ca2+ inflows, meanwhile, PKC can act on the down level protein substrates and promote myocardial hypertrophy. IP3 combines directly with its receptors in sarcoplasmic reticulums and endoplasmic reticulums and causes the release of Ca2+ stored in cellular nuclear envelope gaps to caryoplasms and cytoplasms  is one of the most important reasons besides pressure overloading, nordrenaline and thyrine for myocardial hypertrophy. Ang II, the chief active peptide of RAS, is an effective promoter for cell growth which can facilitate the growth of various cardiovascular cells such as the cardiac myocytes, cardiac fibroblasts and vascular smooth muscle cells , 6. In rt manner . Ang II  and AT2 , 9, and inositol ,5-trisphtoplasms -12. More in-vivo .i.e., extracellular stimulation, intracellular signal transduction and intranuclear activation and rearrangement of genes, which evoke the cells to hypertrophy phenotype changes. Different stimuli of hypertrophy can cause activation of specific genes and form various types of distinctive myocardial hypertrophy , which is the unique protein phosphatase found to be activated by Ca2+ and calmodulin (CaM). CaN is a heterodimer composed of one 61 KD catalytic subunit CnA and one 19 KD regulatory subunit CnB, and generally distributes in a variety of tissues including the heart. It was reported that all the increase of intracellular Ca2+ from different origins could activate CaN. The nuclear factors of activated T cells (NFAT3) could be dephosphorylated by activated CaN, enter the cell nuclei, and act on the zinc finger transcription factor GATA4 to cause hypertrophy of cardiac myocytes and expression of embryo genes (Viewed from cellular and molecular levels, myocardial hypertrophy consists of the following three fundamental aspects, ertrophy , which dertrophy , i.e., Pyo genes -19. Hypeyo genes . This ilyo genes -23. ProtBuckwheat rutin is a major component of buckwheat flavonoids, and rutin exists in no grains other than buckwheat. Previous studies , 24 have


Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+–pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca2+ transporter at the inhibition site of hydroquinones.Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our  The effect was attributed to inhibition of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2+ discharge from intracellular stores in a glioblastoma cell line as occurred under the presence of the high affinity SERCA inhibitor thapsigargin (TG) The irreversible rise of cytosolic Ca2+ leading to cell death by apoptosis. The process was observed in several tumor cell lines including glioblastoma, breast carcinoma, pancreatic carcinoma, Burkitt's lymphoma and multiple myeloma It has been shown that DMC, and in a lesser extent CLX, put in motion the endoplasmic reticulum stress response mediated by sustained elevation of cytosolic free Ca2+-pump from sarco-endoplasmic reticulum, is the main intracellular transporter of most eukaryotic cells involved in the removal of cytosolic Ca2+. It builds up and maintains a >104-fold concentration gradient owing to the uphill transport of Ca2+ from the cytosol to the luminal space of the sarcoplasmic reticulum (SR) 2+ signals that give rise to a wide variety of cellular responses SERCA, also termed CaSince SERCA inhibition unleashes apoptotic cell death, the aim of the present study was to shed light on the effect of DMC as antitumor agent on SERCA catalytic and transport functions. Information on the interaction site of CLX and DMC was also pursued.This study was performed in accordance with the European Union Council Directive of 22 September 2010 (2010/63/EU) and reviewed and approved by the Ethical Committee of the University of Murcia.Streptomyces chartreusensis A23187 from Calbiochem and the Ca2+ standard solution Titrisol were provided by Merck KGaA . 45CaCl2, [γ-32P]ATP and [6-3H(N)]D-glucose were New England Nuclear products from PerkinElmer . The liquid scintillation cocktail Optiphase HiSafe 3 was also from PerkinElmer. HAWP type nitrocellulose filters with 0.45-µm pore size for vacuum filtration were from Merck Millipore . All other analytical grade reagents were obtained from Sigma-Aldrich.DMC was obtained from Sigma-Aldrich  and CLX was from BioVision . 2+ concentrations were adjusted by adding appropriate volumes of CaCl2 and EGTA stock solutions as previously described 2+-EGTA complex 2+ ligands and the pH in the medium. A nominally Ca2+-free medium was established by including excess EGTA without any Ca2+ addition. Free Ca2+ concentration was expressed as negative logarithm of free Ca2+ (pCa). For instance, 1 mM EGTA and 0.686 mM CaCl2 give a pCa value of 6.0.Defined free Ca2+ ionophore A23187 (leaky vesicles) were used. SERCA1a is by far the most abundant protein in the microsomal preparation from rabbit skeletal muscle 2+ sites. The experimental protocols described in this study, i.e., rates of active Ca2+ transport and Ca2+-dependent ATP hydrolysis, Ca2+ binding to the high affinity transport sites and phosphoenzyme (EP) formed from ATP in the presence of Ca2+ have been successfully applied by others for the functional characterization of SERCA using the abovementioned microsomal fraction The starting material was hind leg skeletal muscle from adult female New Zealand rabbit. Right-side vesicles mainly derived from longitudinal tubules of the SR membrane were obtained as described by Eletr and Inesi 2+ accumulation inside native vesicles was measured at 25°C by a radioactive procedure 2, 1 mM EGTA, 0.686 mM [45Ca2+]CaCl2 at ∼2 000 cpm/nmol, 0.02 mg/ml native vesicles and 5 mM potassium oxalate in the absence or presence of a given drug concentration. The transport process was started by adding 1 mM ATP and stopped at different times by filtering under vacuum 0.5 ml aliquots. Thereafter, the filters were rinsed with 5 ml of ice-cold medium containing 20 mM Mops, pH 7.0 and 1 mM LaCl3 and the retained radioactivity was evaluated by liquid scintillation technique.The initial rate of Cai) release was measured at 25°C with a malachite green reagent 2, 1 mM EGTA, 0.686 mM CaCl2, 0.05 mg/ml native vesicles, 5 mM potassium oxalate and 1 mM ATP in the absence or presence of a certain drug concentration. Aliquots of the reaction medium containing 0.02 ml were withdrawn at different time intervals and mixed with 0.8 ml of malachite green reagent to allow color development. Measurements with leaky vesicles were performed in the reaction medium described above but including 4 µM A23187 instead of potassium oxalate. The Ca2+–dependent activity was calculated by subtracting the hydrolytic activity measured in the absence of Ca2+.The initial rate of inorganic phosphate  were filtered under vacuum without any rinsing. Radioactive tracers 45Ca2+ and 3H in the filters were measured by liquid scintillation counting.Equilibrium experiments of Ca2+ in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.2 mM EGTA, 0.138 mM [45Ca]CaCl2 at ∼10 000 cpm/nmol, 0.05 mg/ml native vesicles and 1 mM ATP. When indicated, 50 µM drug, 5 mM EGTA  or 5 mM EGTA plus 50 µM drug was added to the loading medium after 2 min. Then, aliquots of 0.5 ml containing 0.025 mg protein were filtered at different times and the filters were rinsed with 5 ml of cold medium composed of 20 mM Mops, pH 7.0 and 1 mM CaCl2.Native vesicles were actively loaded at 25°C with radioactive Ca32P]ATP in the presence of Ca2+ was measured by the acid quenching technique described by Inesi et al. 32P retained by the filters was evaluated by liquid scintillation counting.Radioactive EP formed from ATP at ∼50 000 cpm/nmol to give a final ATP concentration of 40 µM. The phosphorylation reaction was stopped at different times by adding 5 ml of quenching solution.The initial reaction medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2 mg/ml SR vesicles, 8 µM A23187 and a certain DMC concentration when indicated was distributed in 0.5 ml aliquots. The phosphorylation reaction was started by adding 20 µl of 1 mM [γ-32P]ATP at ∼50 000 cpm/nmol and 17.2 mM CaCl2. Final concentrations after mixing were 40 µM ATP and 0.686 mM CaCl2. The phosphorylation reaction was arrested at different time intervals by adding 5 ml of quenching solution.The initial reaction medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.686 mM CaCl2, 0.4 mg/ml SR vesicles, 16 µM A23187 in the absence or presence of 50 µM DMC. Aliquots of 0.25 ml were mixed with 10 µl of 1 mM [γ-32P]ATP at ∼50 000 cpm/nmol. After 5 s, the reaction mixture was 10-fold diluted with 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.686 mM CaCl2 and 40 µM ATP and the reaction was halted at serial times by adding 2 ml of 438 mM perchloric acid and 7 mM sodium phosphate. Quenched samples were processed as described above.The initial phosphorylation medium was 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2+ was obtained from Protein Data Bank (PDB) ID code 2AGV (http://www.rcsb.org). All non-protein components were deleted from the protein structure and the CLX file in pdb format was obtained from PDB ID code 1OQ5. Avogadro software (http://avogadro.openmolecules.net/) http://autodock.scripps.edu/resources/adt) http://vina.scripps.edu/) http://www.cgl.ucsf.edu/chimera) tert-butyl)-hydroquinone (BHQ) and TG (retrieved from PDB ID code 2AGV) by comparing the initial binding conformation of BHQ in the crystal structure and the docked pose obtained from docking simulations.For protein and ligands processing, the coordinates of the three-dimensional SERCA structure in the absence of CaExperimental results correspond to mean values of at least three independent determinations, each performed in duplicate and standard deviations are indicated by error bars. Curve fitting was performed by non-linear regression using version 11.0 of the SigmaPlot program from Systat Software .2+-pump were initially studied by measuring Ca2+-ATPase activity. To this end, SR vesicles leaky to Ca2+ were exposed to DMC before ATP was added to initiate the catalytic turnover. The initial rate of the Ca2+–dependent ATP hydrolysis was gradually inhibited as the drug concentration was raised and a half-maximum effect was observed at 32 µM  space that prevents Ca2+ transport but does not induce Ca2+ release, so that this type of uncoupling can be ruled out.Uncoupling due to increased membrane permeability will produce rapid Caas added . Rapid Cperative , or arreperative , indicate of DMC . The con2+-free and Ca2+-bound conformations that may or not be phosphorylated are key molecular structures involved in the pumping mechanism. Ca2+ binding to the pump on the cytosolic side triggers a large structural rearrangement 2+ binding  or Ca2+-bound conformation  were exposed to the drug before phosphorylation (2+-free conformation. In other words, the Ca2+-bound conformation was not protected from DMC and therefore DMC rapidly shifted the Ca2+ binding equilibrium retaining the Ca2+-free conformation. Moreover, the mass effect showing partial recovery of the EP level in the presence of DMC and high Ca2+ (2+ binding. These data suggest that the population of transporter with Ca2+-bound conformation during the catalytic turnover in the presence of DMC increases when Ca2+ is raised but does not reach the EP level observed in the absence of inhibitor.Certain pump inhibitors, including TG, exert the effect when exposed to the Carylation . This inigh Ca2+  was cons2+ translocation across the membrane and then hydrolysis of EP to complete the reaction cycle. The kinetics of EP decomposition that is considered a rate-limiting step was not affected by the presence of a DMC concentration producing 60% inhibition of the Ca2+-ATPase activity (Conformational transitions of EP species lead to Caactivity .2+-pump that stabilizes the Ca2+-free conformation. The stabilization involves hydrogen bonds between the two hydroxyl groups of BHQ, one with Asp59 on transmembrane helix M1 and the other with Pro308 on transmembrane helix M4 o bend.BHQ is a transmembrane inhibitor of the SR Cao in the site entrance. The docking pose also reveals altered ligand orientation when the phenyl ring substituent 4-methyl was changed to 2,5-dimethyl, i.e., CLX vs. DMC. This is consistent with the observed difference in the SERCA residues that are involved in the CLX or DMC-protein interaction.DMC is a 1,5-diaryl-substituted pyrazole containing dimethylphenyl and benzenesulfonamide moieties. Molecular modeling applied to CLX and related structures indicated that the terminal phenyl ring and the polar carboxamide or sulfonamide group at the other extreme of the molecules appear approximately perpendicular to each other in the 3D structure 2+-pump inhibition by interfering with Ca2+ binding and uncoupling related with membrane permeabilization activity. Both actions can explain the cytotoxic and anti-proliferative effects that have been attributed to the drug. It remains to be established how in vivo selectivity of DMC on tumor cells is achieved.In conclusion, DMC binding occurs at the BHQ binding site and the effect is a combination of Ca


Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), formation of ceramide, oxidative stress and activation of p38 kinase. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca2+]i and the effect was not dependent on presence of extracellular Ca2+. Piperlongumine significantly increased ROS formation and ceramide abundance. Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+ but at least partially due to ROS and ceramide formation. Moreover, the study explored the involvement of [Ca2+]i, ROS formation and ceramide abundance.To the best of our knowledge, an effect of piperlongumine on suicidal erythrocyte death has never been reported before. The purpose of the present study was to explore whether piperlongumine triggers eryptosis, The present study explored the putative effect of piperlongumine on eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane with subsequent translocation of phosphatidylserine to the cell surface. Phosphatidylserine exposing erythrocytes were identified by annexin-V-binding in flow cytometry. As illustrated in A further hallmark of eryptosis is cell shrinkage. Cell volume was thus estimated from forward scatter in flow cytometry. As shown in 2+ activity ([Ca2+]i). In order to estimate [Ca2+]i, erythrocytes were loaded with Fluo3-AM and Fluo3 fluorescence determined by flow cytometry. As illustrated in Both, cell membrane scrambling and cell shrinkage could have resulted from increase of cytosolic Ca2+. To this end, erythrocytes were exposed for 48 h to 30 µM piperlongumine in the presence or nominal absence of extracellular Ca2+. As shown in 2+, an effect, however, not reaching statistical significance. In the nominal absence of extracellular Ca2+, piperlongumine still significantly increased the percentage of annexin V binding erythrocytes. Thus, the effect of piperlongumine on cell membrane scrambling did not critically depend on Ca2+ entry.Additional experiments explored, whether the piperlongumine induced cell membrane scrambling required entry of extracellular Can = 4), respectively.In order to determine the effect of piperlongumine exposure on hemolysis, the percentage of hemolysed erythrocytes was quantified from hemoglobin concentration in the supernatant. According to the hemoglobin concentration, a 48 h incubation with 0, 5, 10, 15 and 30 µM piperlongumine resulted in hemolysis of 2.4% ± 0.4%, 2.6% ± 0.3%, 2.5% ± 0.5%, 2.5% ± 0.2% and 4.6% ± 1.5%  were determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). As shown in 2+]i by ceramide, additional experiments explored, whether piperlongumine increases ceramide formation. The abundance of ceramide at the erythrocyte surface was visualized with an anti-ceramide antibody. As shown in As cell membrane scrambling could be further elicited even at constant i), a major trigger of eryptosis [Piperlongumine did not significantly increase cytosolic Caryptosis . Insteadryptosis . In tumoryptosis ,15,16,17The phosphatidylserine exposing erythrocytes attach to respective receptors of phagocytes with subsequent engulfment and degradation of the defective erythrocytes . Thus, eExcessive eryptosis may become pathophysiologically relevant. The clearance of phosphatidylserine exposing erythrocytes from circulating blood may lead to anemia as soon as the loss of eryptotic erythrocytes fails to be outweighed by similar formation of new erythrocytes . TreatmeThe sensitivity of erythrocytes to piperlongumine may be augmented by additional exposure to further eryptosis triggering xenobiotics ,78,79,804, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid , 5 glucose, and 1 CaCl2. For the experiments, erythrocytes were incubated in vitro at a hematocrit of 0.4% at 37 °C for 48 h. Where indicated, erythrocytes were exposed to piperlongumine  at the indicated concentrations. In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). The blood was centrifuged at 120 rcf for 20 min at 23 °C and the platelets and leukocytes-containing supernatant were disposed. Erythrocytes were washed in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur. The adequacy of Fluo-3 fluorescence as a tool to estimate cytosolic Ca2+ activity has been tested in several previous studies [After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaCl studies ,92,93,94ROS production was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) . BrieflyFor the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 µg/mL anti ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.For the determination of hemolysis, the samples were centrifuged  after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.t test as appropriate. The number of different erythrocyte specimens studied is given as n. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and In conclusion, the present paper unequivocally and significantly demonstrates that piperlongumine stimulates eryptosis with erythrocyte shrinkage and cell membrane scrambling. Thus, piperlongumine is able to trigger suicidal death in cells lacking mitochondria and nuclei. The cellular mechanisms involved include oxidative stress and ceramide formation. To the best of our knowledge, an effect of piperlongumine on suicidal erythrocyte death or of ceramide formation in any cell type has never been reported before.


Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca2+ uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca2+ channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca2+ signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca2+ rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca2+ uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca2+ rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca2+ concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.Mitochondrial Ca In 2011 a 35-kDa protein, which was initially referred to as CCDC109A and then renamed mitochondrial Ca2+ uniporter (MCU) was described to show many if not all of the typical characteristics of a Ca2+ uniporter52+ channels. Interestingly, these MCU oligomers are part of larger protein complexes of the inner mitochondrial membrane, indicating the existence of complex mitochondrial Ca2+ uptake machineries that include other regulatory subunits91012132+ uptake, have not been found in MCU containing protein complexes17192+-binding domains and forms large complexes of app. 480 kDa17162+-binding EF-hand domains of MICU1 can undergo large conformational changes upon binding of Ca2+222+ concentration ([Ca2+]mito) at low resting Ca2+ levels while it is thought to facilitate mitochondrial Ca2+ uptake upon high Ca2+ concentrations18242+ MICU1 exists as a hexamer, while Ca2+ binding to the two EF-hands results in a rearrangement to MICU1 dimers2+ channel activity that gets released upon (re-)organization of MICU1 hexamers/multimers2+-dependent rearrangements of MICU1 multimers indeed occur and regulate the activity of MCU in intact cells, and the impact of MCU and EMRE to the structural (re-)organization of MICU1 have not been investigated so far. Hence, the kinetics of the rearrangement of the MICU1 multimers upon cytosolic Ca2+ elevation and whether or not the mitochondrial matrix Ca2+ concentration or membrane potential (ψmito) have an impact on the structural reorganization of MICU1 remain elusive.While the phenomenon of mitochondrial membrane potential-dependent Ca2+ signals as well as ψmito. Simultaneous measurements of cytosolic Ca2+ with either mitochondrial Ca2+ signals or MICU1 rearrangement were performed to verify whether MICU1 multimers indeed retard MCU activation in intact cells. Finally, we tested whether the Ca2+-sensitive rearrangement of MICU1 is dependent of the expression level of MCU and EMRE. Our data provide new insights in the dynamics, regulation, and molecular effect of the structural (re-)organization of MICU1 that adds to our current understanding of the complex molecular mechanisms of MCU activation.In this study we used a FRET-based live-cell imaging approach to dynamically monitor the kinetics of the structural (re-)organization of MICU1 and to explore its dependence from cytosolic and mitochondrial Ca2+-mobilization by an inositol 1,4,5-trisphosphate- (IP3-)generating agonist affects the arrangement of MICU1 in intact cells, Förster energy transfer (FRET) imaging was applied in cells co-expressing MICU1 fused to either cyan fluorescent protein (MICU1-CFP) or yellow fluorescent protein (MICU1-YFP). Because MICU1 has been shown to assemble in hexamers in the absence of Ca2+ and rearranges to dimers upon Ca2+ binding2+ and its subsequent binding to MICU1 should yield disaggregation of MICU1 hexamers and reduce the inter-MICU1 FRET signal. Indeed, cell treatment with the IP3-generating agonist histamine2+ levels. In the nominal absence of extracellular Ca2+, the histamine-triggered decrease of inter-MICU1 FRET was more transient and slowly developed upon subsequent addition of extracellular Ca2+  by the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor 2,5-di-tert-butylhydroquinone (BHQ)2+ uptake in response to cell treatment with BHQ was largely increased in cells treated with the 3´UTR siRNA against MICU1  to trigger rearrangement of the MICU1 multimers of 4.4 (3.7–5.2) μM in HeLa cells  in combination with the near ultra-violet excitable cytosolic Ca2+ sensor fura-2 was performed in order to compare the kinetics of cytosolic and mitochondrial Ca2+ signals. This approach revealed a lag time (ΔT) of 1.50 ± 0.08 s between the cytosolic Ca2+ rise and its transition into the mitochondrial matrix upon intracellular Ca2+ mobilization by the IP3-generating agonist histamine   .2+ signals, we imaged dynamic changes of MICU1 FRET simultaneously with cytosolic Ca2+ in response to histamine  four sequential steps: (i) cytosolic Ca2+ elevation and transfer into the intermembrane space, (ii) binding of Ca2+ to MICU1 oligomers, (iii) MICU1 de-oligomerization, and (IV) activation of the MCU.In order to visualize how the temporal pattern of MICU1 de-oligomerization refers to cytosolic Caistamine . This ap+ uptake . Notablychondria . These dmito) impacts rearrangement of MICU1 multimers, cells were treated with oligomycin and the uncoupling agent carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) to efficiently depolarize mitochondria2+ signals in response to histamine were observed organization. Our data highlight that an elevation of cytosolic free Ca2+ rearranges MICU1 multimers to smaller complexes in intact cells. These findings are consistent with a recent report showing that recombinant Ca2+-free MICU1 exists as hexamer and rearranges in the presence of Ca2+ to dimers72+ uptake at low cytosolic Ca2+ is in line with previous reports about the inactivity of MCU under resting conditions8The present study describes the dynamics of MICU1 (re-)organization in response to cytosolic Cafirst, the high expression of FP tagged MICU1 proteins might result in lower oligomerization states, and second, upon histamine-induced intracellular Ca2+ release, MICU1 de-oligomerization most likely occurs predominantly within the junctions between the ER and mitochondria where high Ca2+ gradients are developed2+.Our approach of live-cell monitoring the inter-MICU1 FRET allows the visualization of MICU1 (re-)organization within the intact cell in life-time. Notably, calculation of the changes in MICU1 FRET probability in the transition from hexamers to dimers revealed a theoretically achievable reduction of FRET by app. 52% . However2+ dynamics data revealed that the rearrangement of MICU1 multimers by cytosolic Ca2+ strictly correlates with the cytosolic Ca2+ concentration and is rapidly reversible. The in situ calibration indicates that the rearrangement of MICU1 senses Ca2+ changes in the range between the low basal Ca2+ levels of 100 nM up to 100 μM. With an EC50 of around 4 μM, the Ca2+ sensitivity of MICU1 rearrangement lays exactly in the range of Ca2+ hot spots (3.78 and 16.4 μM) that have been measured at the outer mitochondrial membrane between the junction of mitochondria and the ER3-mediated intracellular Ca2+ mobilization almost instantly yielded MICU1 rearrangement, while Ca2+entry via the store-operated Ca2+ entry pathway312+ release and entering Ca2+ are consistent with previous reports that described a rather slow on-kinetics of entering Ca2+ at the mitochondrial surface without the formation of Ca2+ hot spots and slow mitochondrial Ca2+ accumulation1427Our approach that allowed correlation of the kinetics of MICU1 (re-)organization with cellular Ca2+, the inhibition of SERCA by BHQ yielded only slow and small rearrangement of MICU1 multimers and, thus, explains the marginal effect of SERCA inhibition on mitochondrial Ca2+ uptake. Since the diminution of MICU1 expression substantially gains mitochondrial Ca2+ uptake in response to intracellular Ca2+ release by SERCA inhibition, the prominent role of MICU1 as a negative regulator of mitochondrial Ca2+ uniport is clearly demonstrated. This assumption is further supported by our experiments in which cytosolic and mitochondrial free Ca2+ were simultaneously measured and the lag time of app. 1.5 s between cytosolic Ca2+ elevation and the mitochondrial Ca2+ uptake was strongly reduced in cells with diminished expression of MICU1. Hence, the correlation between cytosolic Ca2+ elevation, the rearrangement of MICU1 multimers and mitochondrial Ca2+ signals, revealed that MICU1 reorganization temporally occurs in between the Ca2+ rise within the two compartments. Notably, significant mitochondrial Ca2+ uptake appears to occur at app. 50% rearrangement of MICU1 multimers. These data demonstrate that for activation of the MCU/EMRE complex to achieve mitochondrial Ca2+ influx, app. 50% of the MICU1 multimers have to be rearranged. Although these findings might be due to the overexpression of MICU1 in our model, the MICU1-dependent lack of mitochondrial Ca2+ uptake in response to SERCA inhibition supports this assumption.Similar to entering Ca2+ uptake strongly depends on Ψmito that establishes the driving force for Ca2+ uniport into the organelle. However, whether or not Ψmito also impacts on MICU1 organization has not been investigated so far. Our data with completely depolarized mitochondria revealed an unchanged MICU1 dynamics despite a greatly reduced mitochondrial Ca2+ uptake. These data demonstrate that neither Ψmito nor matrix Ca2+ are involved in the rearrangement of MICU1 complexes and confirms cytosolic Ca2+ as possible the sole regulator of MICU1 (re-)organizationMitochondrial Ca2+ uptake, the Ca2+-triggered rearrangement of MICU1 multimers, their arrangement upon the reduction of cytosolic Ca2+ to basal levels, and the sensitivity to cytosolic Ca2+ remained unaffected by the reduction of MCU or EMRE. These data demonstrate that the structural (re-)organization of MICU1 upon elevation of cytosolic free Ca2+ does not involve MCU or EMRE indicating that the Ca2+-induced rearrangement of MICU1 multimers is a robust process that does most likely not require other interaction partners.Considering the interaction of MICU1 with EMRE and MCU716181923251628first, MICU1 FRET rearrangement follows cytosolic but not matrix mitochondrial Ca2+, second, MICU1 knockdown results in a faster mitochondrial Ca2+ uptake, third, rearrangement of MICU1 FRET precedes mitochondrial Ca2+ uptake, forth, MICU1 FRET is independent from Ψmito, and fifth, neither knockdown of MCU nor of EMRE influences MICU1 FRET rearrangement indicate that MICU1 anchors with its N-terminus in the IMM while the core protein is oriented towards intermembrane space and not to the matrix.Despite the topology of MICU1 is still under debate1821252+ uptake. For the first time, the kinetics and adjustments of one of the most important molecular gatekeeper of mitochondrial Ca2+ uptake was visualized in intact cells. Our data revealed cytosolic Ca2+ as the most prominent regulator of the structural organization of MICU1, which in the form of a homo-multimere potently inhibits the MCU/EMRE mitochondrial Ca2+ channel complex. Moreover, neither Ψmito nor matrix Ca2+, nor MCU or EMRE were found to affect the Ca2+-controlled (dis)assembly of MICU1 multimers. Finally, our data provide important details for a better understanding of the molecular regulation of an intricate mitochondrial Ca2+ uptake machinery.Our results provide new mechanistic insights in the regulation of mitochondrial Ca2 135 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 D-glucose, 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin pH 7.4. For Ca2+ measurements HeLa cells were perfused in a HEPES buffered solution containing (in mM): 2 CaCl2, 140 NaCl, 5 KCl, 1 MgCl2, 1 HEPES and 10 D-Glucose pH 7.4. To achieve a nominal Ca2+ free environment either 1 mM EGTA was added instead of 2 mM CaCl2. For Kd determination of MICU1 FRET we used the same buffer containing 3 mM EGTA supplemented with 3–10 μM ionomycin  and CaCl2 was added according to the CaBuff software  to obtain buffer solutions of 0.1, 1, 10, 100 and 1000 μM free Ca2+ concentrations.All reagents and chemicals for buffers and solutions were obtained from Carl Roth . Fura-2/AM was obtained from Teflabs . BHQ and histamine were purchased from Sigma-Aldrich . FCCP and oligomycin were from Abcam Biochemicals . Prior to experiments HeLa cells were stored and loaded with fura-2/AM in a buffer composed of (in mM): 2 CaCl2/95% air). 2–3 days prior experiments HeLa cells were transfected in DMEM (without FCS and antibiotics) with plasmids  and/or siRNA (100 nM) using 4 μg/ml TransFastTM transfection reagent .HeLa cells at passage >50 were cultured on glass cover slips (Ø = 30 mm) using DMEM  containing 10% FCS , penicillin (100 U/ml) and streptomycin (100 U/ml) in a humidified incubator  of human MICU1  without a stop codon was amplified from a HeLa cDNA by PCR using primers : forward: 5′-ACGGATCCACCATGTTTCGTCTGAACTCAC-3′ and reverse: 5′-ACGAATTCCTGTTTGGGTAAAGCGAAGTCC-3′. The PCR fragment was cloned in a pcDNA3.1 (+) vector via BamHI and EcoRI sites. The cds of either enhanced cyan fluorescent protein (CFP) or citrine (YFP) were amplified with the same primers: forward: 5′-AAGAATTCATGGTGAGCAAGGGCGAGGAG-3′ and reverse: 5′-CCTCTAGAACTTGTACAGCTCGTCCATGC-3′ and each C-terminally fused to hMICU1 using the EcoRI and XbaI sites. EF hand mutated MICU1 and O-GECO1 constructs were purchased from Addgene . The EF hand mutated MICU1 (EFmut) was amplified using the same primers as described above and C-terminally fused with either ECFP or Citrine like the wild-type MICU1. A mitochondrial targeted mtO-GECO1 was generated upon PCR amplification of O-GECO1 using the forward primer 5′-AACTCGAGTATGGTCGACTCATCACGTCG-3′ and a reverse primer 5′-GCAAGCTTTTACTTCGCTGTCATCAT-3′ and the PCR fragment was N-terminally fused with the mitochondrial targeting sequence (4mt) via XhoI and HindIII restriction sites in a pcDNA3.1(–) vector. 4mtD1GO-Cam was constructed as previously describedTo determine hEMRE and hMCU silencing HeLa cells were transiently transfected with the respective siRNA(s). The PEQLAB total RNA isolation kit  was used for total RNA isolation and 1 μg of each RNA sample were reverse transcribed with a cDNA synthesis kit . The efficiency of siRNA mediated knockdown was validated by real time PCR on a LightCycler 480 . Target genes and human GAPDH  as housekeeping gene were amplified within the respective cDNA samples using the GoTaq® qPCR Master Mix (Promega) and specific real time primer pairs (Invitrogen): hMCU forward: 5′-TTCCTGGCAGAATTTGGGAG-3′, hMCU reverse: 5′-AGAGATAGGCTTGAGTGTGAAC-3′; hEMRE forward: 5′-TCGCTGGCTAGTATTGGCAC-3′, hEMRE reverse: 5′-GGAGAAGGCCGAAGGACATT-3′. Relative expression of the hEMRE and hMCU were normalized to GAPDH expression and data were analyzed by the REST software .2+ imaging and dynamic FRET measurements between MICU1-CFP and MICU1-YFP were performed on an inverted microscope  equipped with a polychromator illumination system  and a thermoelectric-cooled CCD camera . Cells were imaged with a 40× oil-immersion objective (Zeiss). Fura-2 and the 4mtD1GO-Cam were alternately excited at 340 nm or 380 nm and at 477 nm, respectively, with an ultra-fast switching monochromator, the Polychrome V (Till Photonics), equipped with an excitation filter (E500spuv) and a dichroic filter . Emitted light was simultaneously collected at 510 nm (Fura-2 and GFP of GO-Cam) and at 600 nm (FRET-channel of GO-Cam) using a single beam splitter design  that was equipped with a dual band emission filter  and a second dichroic filter . Images were recorded with a CCD camera . The digital imaging system was controlled by the live-acquisition software v2.0.0.12 (Till Photonics), as described previously. For the dynamic CFP-YFP FRET measurements transfected cells were excited at 440 ± 10 nm , and emission was recorded at 480 and 535 nm using emission filters  mounted on a Ludl filterwheel. Results of FRET measurements are shown as the ratio of FFRET/FCFP or as (FFRET/FCFP)/R0 , to correct for photobleaching and/or photochromism, as described previously2+]cyto was performed in HeLa cells co-transfected with MICU1-CFP, MICU1-YFP and O-GECO1 or mtO-GECO1, respectively. MICU1-CFP and (mt)O-GECO1 were alternately illuminated at 430 nm and 575 nm, respectively, in single individual cells. Emitted light was collected at 535 nm for MICU1 FRET and 610 nm for (mt)O-GECO1 using the XF56 filter set of Omega Optical.CaU test was applied. Differences in means among multiple data sets were analyzed by using one-way analysis of variance (ANOVA) and subsequent Bonferroni post hoc test and two-tailed Student’s t-test. P < 0.05 was considered significant. Data were analyzed either with MS Excel 2011 or with Graphpad Prism version 5.0.All experiments were repeated at least three times on two different days. Data from multiple experiments were quantified to determine absolute or percent changes, expressed as mean ± SEM where n reflects the actual number of repeats. Statistical analyses were performed with unpaired Student’s t-test. When not normally distributed, a Mann-Whitney How to cite this article: Waldeck-Weiermair, M. et al. Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+. Sci. Rep.5, 15602; doi: 10.1038/srep15602 (2015).