ATP channels, depolarization of the plasma membrane, influx of calcium and the eventual secretion of insulin. Such mechanism suggests that beta-cell metabolism should have a functional regulation specific to secretion, as opposed to coupling to contraction. The goal of this work is to uncover contributions of the cytoplasmic and mitochondrial processes in this secretory coupling mechanism using mathematical modeling in a systems biology approach.Pancreatic beta-cells respond to rising blood glucose by increasing oxidative metabolism, leading to an increased ATP/ADP ratio in the cytoplasm. This leads to a closure of K1F0 ATPase. Redox shuttles and mitochondrial Ca2+ handling were also modeled.We describe a mathematical model of beta-cell sensitivity to glucose. The cytoplasmic part of the model includes equations describing glucokinase, glycolysis, pyruvate reduction, NADH and ATP production and consumption. The mitochondrial part begins with production of NADH, which is regulated by pyruvate dehydrogenase. NADH is used in the electron transport chain to establish a proton motive force, driving the F2+ and other metabolic parameters in response to changes in substrate delivery at steady-state and during cytoplasmic Ca2+ oscillations. Our analysis of the model simulations suggests that the mitochondrial membrane potential should be relatively lower in beta cells compared with other cell types to permit precise mitochondrial regulation of the cytoplasmic ATP/ADP ratio. This key difference may follow from a relative reduction in respiratory activity. The model demonstrates how activity of lactate dehydrogenase, uncoupling proteins and the redox shuttles can regulate beta-cell function in concert; that independent oscillations of cytoplasmic Ca2+ can lead to slow coupled metabolic oscillations; and that the relatively low production rate of reactive oxygen species in beta-cells under physiological conditions is a consequence of the relatively decreased mitochondrial membrane potential.The model correctly predicts changes in the ATP/ADP ratio, Caβ-cells.This comprehensive model predicts a special role for mitochondrial control mechanisms in insulin secretion and ROS generation in the beta cell. The model can be used for testing and generating control hypotheses and will help to provide a more complete understanding of beta-cell glucose-sensing central to the physiology and pathology of pancreatic  Pancreatic β-cells are adapted to sense blood glucose and other secretagogues to adjust insulin secretion according to the needs of the organism. Rather than activating specific receptor molecules, glucose is metabolized to generate downstream signals that stimulate insulin secretion. Pancreatic β-cells respond to rising blood glucose by increasing oxidative metabolism, leading to increased ATP production in mitochondria and in an enhanced ratio of ATP to ADP (ATP/ADP) in the cytoplasm c versus glucose concentration  ratio was found in rat islets m and oxygen consumption were also observed with glucose stimulation in control INS-1 cells [Several studies have confirmed an increase in the ATP/ADP ratio in response to high glucose  and O2 consumption increased simultaneously with Ψm in our simulation of the β-cell . The model offers the following explanation of this contradiction. In our model (as in living cells) the electron transport rate (Equation 5) depends on at least two factors: one is a decrease in the electron transport rate with an increase in Ψm (Equation 5C) but another factor is an increase of this rate with increased substrate concentration (NADH) (Equation 5A). Increasing the electron transport rate simultaneously with Ψm means that the enhancement of Jhres as a result of the increased [NAPH]m was greater than its decrease with the rise of Ψm following the step increase in glucose. Substrate concentrations are usually maintained at constant or saturated levels in experiments with isolated mitochondria, where one can only see inhibition of the electron transport rate with increased Ψm.1. A basic principle of mitochondrial energetics is given by the inverse relationship between the respiratory flux and Ψion rate ,56. We sc. This mechanism corresponds to Equation 7A in our model. Experimentally this hypotheses has been tested in permeabilized clonal β-cells, where ATP/ADP ratios can be externally fixed showing that a decrease in [ADP] led to decreased O2 consumption c following a step increase in glucose. As a result, ATP synthesis and respiration rate increase despite decreased [ADP]c and the ATP/ADP ratio increased with a step glucose increase . A decrease in the free [ADP]β-cell regulatory mechanisms endow this cell type with unique metabolic properties to control insulin secretion in comparison with metabolism in other cell types. For example, liver cells maintain a stable ATP/ADP equilibrium while respiring at widely varying rates c was also set to an elevated constant level (700 mM), that was 5-fold higher than the calculated [ADP]c level (at 9 mM glucose) in β-cells.Specific  β-cells ,25. We e β-cells ,62. To sme) was increased in steps. Mitochondrial F1F0 ATPase activity (Vmph) was unchanged. Simulated Ψm and the rate of ATP production (Jph) were significantly increased with an increased Vme, such that F1F0 ATPases work with maximal activity under these conditions  as well as by the increased [ADP]c in simulated muscle cells in comparison with β-cells. Note that the rate of ATP production (Jph) depended only slightly on Ψm change when Ψm was increased above 160 mV, since these levels of Ψm were saturating for F1F0 ATPase activity  magnitude was significantly lower in the muscle cell simulation in comparison with respiration rate  at increased Vme even with invariant coefficients for the proton leak, since the rates of respiration and ATP production were highly increased but a coefficient of leak (Jh1) would remain as constant  was significantly lower in pancreatic islets even in high glucose, compared with brain or heart , supportm . We simulated how changes in leak activity affect the response of the variables. To do this we increased the regulated coefficient of proton leak  threefold leading to an increase in the total proton leak rate by twofold  we set the regulated proton leak coefficient equal to zero in Equation 8 (P1r = 0)  Figure . In this2+]c, altering cellular sensitivity to glucose challenge. This effect of proton leak is only possible when the ATP/ADP ratio can be regulated by changes in Ψm, i.e. when Ψm lies below the β-cell maximal level for ATP production. On the other hand, in muscle cells Ψm can be maintained at a high level (see above) and changes in Ψm exert an insignificant effect on the ATP production rate.These simulations show that proton leak can modulate GSIS by shifting the dependence on glucose of the ATP/ADP ratio and c. However, our simulation also shows a novel aspect of this problem: an ability of uncoupling proteins (and other uncoupling agents) to shift the glucose dependence of the ATP/ADP ratio and [Ca2+]c. This shifting mechanism is not simply varying the rates of a few processes. In our model, the shifting mechanism is generally characterized by small changes in Ψm, ATP/ADP ratio or [Ca2+]c at low and high glucose levels. However, larger changes of [Ca2+]c can be expected in the region of physiological glucose concentrations  [c/[NAD+]c .Since duction) ,14,15, wm, Ψm, ATP/ADP ratio as well as [Ca2+]c in response to increased glucose. This was all a consequence of the increased fraction of the glycolytic flux that was directed to lactate production.We also simulated a rise in LDH activity Figure . This le2+]c concentration is already saturated and increased Ψm can lead to increased ROS production (see below).Our simulations confirm that low levels of LDH expression in insulin-secreting cells are important for the correct channeling of pyruvate towards mitochondrial metabolism see ,16). How. How16])β-cells diminished their response to glucose as measured by mitochondrial NAD(P)H, Ψm, cytosolic free ATP and Ca2+ and led to a right shift in the glucose response of insulin secretion c/[NADH]c ratio can vary from about 700:1 to 200:1 in rat liver [+]c/[NADH]c in Cos7 cells and found a ratio of 644. Patterson et al. [+]c/[NADH]c ratio level . NADH generated by glycolysis was efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase in basal conditions in our model H/NAD(P)+ ratio ,42,53,752+]c quickly reached saturation with increased shuttle activity from the basal level. However, a decrease in transport rate coefficient (TNADH) resulted in a decrease in ATP/ADP ratio and in [Ca2+]c from the apparent threshold . The corresponding reduction in pyruvate concentration decreased mitochondrial ATP production, leading to a decreased ATP/ADP ratio and decreased [Ca2+]c. However, glucose consumption did not decrease significantly, because excess glucose influx was shifted to accelerated lactate production in the cytoplasm, as a consequence of the sharp increase in the cytoplasmic NADH concentration  (which would effectively eliminate the glycerol-phosphate shuttle), when the malate-aspartate shuttle was blocked by inhibiting aspartate aminotransferase by aminooxyacetate, glucose-induced increases in cellular ATP content were impaired and insulin secretion was eliminated, whereas the glycolytic flux remained unchanged . However2+]c in mouse islets in the presence of 12 mM glucose c, Ψm and [NADH]m increases and decreased lactate output (JLDH) in Figure NADH was increased from the basal level.The effects of agents known to increase shuttle activities were also examined. Shuttle agonists increased the average m levels are relatively low. Calibrated resting [Ca2+]m in rat islets was approximately 250 nM at 3 mM external glucose. Increasing glucose to 16 mM resulted in a rise of cytoplasmic and mitochondrial [Ca2+] above its resting level [2+]m versus glucose concentration for steady-state  leading to a "push" of electrons through the respiratory chain. This accelerates ATP production by generating more proton motive force, leading to increased [NADH]m and stimulating oxidative phosphorylation m in a control of β-cell oxidative metabolism under physiological conditions is questionable. The initial mitochondrial NADH and Ψm response precedes increased cytoplasmic and mitochondrial Ca2+ in response to a sharp increase in glucose [2+]m increase . For thmediates ,52. Undeors (see ).2+]m can be regulated by mitochondrial Na+/Ca2+ exchanger activity, inhibition of the Na+/Ca2+ exchanger was suggested as a possible target to increase [Ca2+]m and thereby improve insulin secretion in type 2 diabetes. An inhibitor of the exchanger (CGP37157) was shown to prolong mitochondrial Ca2+ signals and increase insulin secretion [2+ channels at high concentrations [Because [Caecretion . (Howevetrations ).2+]m changes on GSIS. The results of simulations .Our model allows an evaluation of the influence of [Cas Figure  showed tc Figure . We foun2+]m due to a large increase in the maximal velocity of Na+/Ca2+ exchange led to an inhibition of ATP production and a decreased ATP/ADP ratio and [Ca2+]c . A high mitochondrial Ca2+ influx and efflux equivalent to an increased proton leak would cause a fall in membrane potential. This would shut down ATP production by the F1F0 ATPase, a process referred to as "short circuiting" in the several mathematical models for β-cell mitochondria in c does not decrease Ψm . The overestimation of the role of Ca2+ fluxes in the dissipation of Ψm in β-cell models c under physiological conditions.Caeostasis ,10,92,93sis see ,26). The. The2+ il volume  and its ee Refs. ,5,92). GReduction in mitochondrial metabolism and/or cellular content (number or volume) can in principle underlie progression to the decreased insulin secretion typical of Type 2 diabetes ,37,94. Fme)  to simulate of the results of experiments where the electron transport chain activity was decreased .We varied the maximal rate of ETC proton pumping , Ψmfailed to hyperpolarize in response to glucose and ATP production and insulin secretion were significantly decreased c and ATP/ADP ratio. In contrast, glucose utilization was only insignificantly decreased.These simulations correspond to data -97 obtaiecreased  found Et1F0 ATPase activity (Vmph)  to simulate of the results of experiments where this activity was changed. Suppression of F1F0 ATPase activity resulted in decreased of ATP synthesis in mitochondria. This decreased [ATP]c and increased [ADP]c leading to a decreased ATP/ADP ratio and decreased [Ca2+]c  (simulations not shown) and insignificant [NADH]m accumulation. Interestingly, here as well as in first case, the rate of glucokinase reaction was not changed because the NADH not utilized for ATP production was expended on increased leak due to increased Ψm (not shown).We varied also the maximal Fc Figure . Decreasm is increased in BHE/cdb rat islets due to increased oxygen radical formation c  displayed severe mtDNA depletion, deficient oxidative phosphorylation and abnormally enlarged islet mitochondria c at the tested glucose levels. This initial increase of [Ca2+]c is due to increased ATP production (and an increase in ATP/ADP ratio) as a result of decreased lactate production  at low and medium glucose levels. This is a result of increased total leak activity that occurs in the model as a consequence of increased mitochondrial content. According to the simulation, lactate production was higher and relative leak was lower (compared with electron transport rate) in basal conditions at high glucose content. For this reason [Ca2+]c did not decrease with increased mitochondrial activity or content at high glucose level ([Glu] = 20 mM)  Figure .2+]c response to glucose, together giving decreased glucose sensitivity  leads to decreased ATP production, ATP/ADP ratio and c oscillations can be independently established from mitochondrial processes when the ATP/ADP ratio achieved some threshold leading to initial depolarization of the plasma membrane [2+]c in the cytoplasm  , where [Ca2+]c increased sharply and the quiescent time was longer than the period with increased [Ca2+]c  Figure . Using tre shown ,23,106).s Figure . This si2+]c changes to activation of metabolic oscillations. Each [Ca2+]c increase during oscillations leads to increased cytoplasmic ATP consumption . This decreases [ATP]c and increases [ADP]c . Increasing the rate of proton flux through F1F0 ATPase led to a decrease in Ψm H oscillations were small and preceded those of calcium by about 0.1 of a period [2+]c periodic maxima  were si2+] oscillations, and oxygen consumption rate and [Ca2+]c changes were approximately in phase. The mechanism of this phenomenon is as yet unknown. However, these data are in accord with our model simulation .Mechanisms other than [ADP]2+ were in response to glucose elevations, presumably tracking oscillations in [Ca2+]c [2+]m followed [Ca2+]c oscillations with a delay of approximately 14 sec [2+]c and [Ca2+]m (14 sec) was simulated in Figure These results of simulations were consistent with observations demonstrating that oscillations in mitochondrial Ca [Ca2+]c ,108,109.y 14 sec ,109. A s2+]c during oscillations results in a decrease in ATP/ADP ratio due to increased ATP consumption [2+]cdecreases, ATP production outweighs ATP consumption leading to an increasing ATP/ADP ratio. However, this pathway is unlikely to be a possible pacemaker mechanism for [Ca2+]c oscillations as has been proposed m follows [Ca2+]c with a particular delay (see above). Our dynamic simulations also clearly show that the independent [Ca2+]c oscillations lead to simulation of [Ca2+]m, Ψm, mitochondrial NADH and respiration oscillations that were similar to experimental observations. These experimental data and our simulations suggest that independent [Ca2+]c oscillations can be a pacemaker in the generation of oscillations of mitochondrial and cytoplasmic parameters in β-cells.However, they have a typical dynamic that is intrinsic to two successive components where c sensitivity to glucose.We found that the adaptive mechanisms in mitochondria can include decreased respiratory activity compared with Fβ-cell energetics permits testing of bioenergetic control hypotheses, provides a basis for further refinement of such steps as oxidative phosphorylation, mitochondrial Ca2+ handling and allow for future incorporation of other biochemical pathways.This comprehensive model of The reaction network of the model is shown in Figure β-cells or islets we have modified some of these parameters to more closely reflect the complex experimental measurements in vivo, where indicated.The parameters for the model are given in Tables β-cells take up glucose by means of a glucose transporter, GLUT2 and/or GLUT1. The uptake is rapid and believed to effectively equilibrate the extracellular and intracellular concentrations of the sugar  is extracellular D-glucose concentration, [ATP]c is the cytoplasmic ATP concentration, KmATP is the Michaelis-Menten constant, Kmgl is the glucose concentration at which the reaction is half maximum, and hgl is the Hill coefficient.where +-dependent step. The flux through glyceraldehydes 3-phosphate dehydrogenase (Jgpd) was assumed to follow irreversible Michaelis-Menten kinetics, where a dependence on [NAD+]c/[NADH]c was written as suggested previously  is the cytoplasmic glyceraldehydes 3-phosphate concentration, KmGPD is the Michaelis-Menten constant, [NAD+]c and [NADH]c are the cytoplasmic NAD+ and NADH concentrations, and KGNc is the [NAD+]c/[NADH]c ratio that gives half maximal glycolytic flux and NADH production.where VJLDH) was described  is the cytoplasmic pyruvate concentration, KmLD is the Michaelis-Menten constant, KLNC is the [NADH]c/[NAD+]c ratio that gives half lactate and NAD+ production. Basal level of VmLDH was evaluated  as the product of several regulated factors:The cytoplasmic pyruvate concentration is the key factor for mitochondrial ATP synthesis. After being transported into the matrix of mitochondria, pyruvate is decarboxylated in a process catalyzed by the multienzyme complex pyruvate dehydrogenase (PDH), thus forming the intermediate acetyl-coenzyme, or carboxylated by pyruvate carboxylase to form oxaloacetate (anaplerotic pathway) ,121. As  prevail . Calcium prevail . We descwheremPDH represents the maximal PDH activity, fPYR is the pyruvate kinetic factor, [NAD+]m and [NADH]m are the mitochondrial NAD+ and NADH concentrations, fPNAD is the [NAD+]m/[NADH]m kinetic factors and FPCa is the calcium activity factor, and [Ca +]m is the mitochondrial Ca + concentration.VPYR and fPNAD are based on the Equation 18 from c is the concentration of free cytoplasmic MgADP, KmADP is the activation rate constant, and hph is the Hill coefficient. AT is the thermodynamic potential factor, where Kph is the membrane potential yielding half maximal ATP production, hp is the Hill coefficient. ACa is a sum of kinetic factors describing the effect of calcium concentration, where KPCam is the [Ca +]m binding constant.VD on free MgADP in medium that can be calculated using the Hill equation, where the mitochondrial oxidative phosphorylation rate increases with increased free MgADP concentration. The apparent half-saturated concentration is in the range of 20 - 45 μM and the Hill coefficient is about 2 c is the cytoplasmic Ca2+ concentration, αm, and αi are the mitochondrial and extramitochondrial activity coefficients.where β-cells, the main mechanism of Ca2+ extrusion from the mitochondria is the Na+/Ca2+ exchanger c and [Na+]m are the cytoplasmic and mitochondrial Na+ concentration, kNaj is the binding constant for sodium and KCaj is the binding constant for Ca2+.2+ uniporter and Na+/Ca2+ exchanger maximum activity from experimental data obtained for living cells. Delay was observed between changes in the cytosolic Ca + concentration and corresponding changes in [Ca2+]m [2+]m on [Ca2+]c changes. We have modeled this delay by setting the maximal activities of the Ca2+ uniporter and the Na+/Ca2+ exchanger at levels that replicate experimental slow [Ca2+]m dynamics (see text).We set Ca [Ca2+]m ,109. Thic and free [ADP] and the mitochondrial variables: [NADH]m, [Ca +]m and Ψm- All fluxes were expressed as mass fluxes per unit time per unit total cell volume.Finally, based on the above relations we determined the dynamic equations for cytoplasmic [G3P], [Pyr], [NADH]The balance equation for the G3P concentration isgpd is the rate constant of G3P consumption in cytoplasm, Vi is the relative cytoplasmic fraction of total cell volume. Coefficient two in the numerator indicates that the breakdown of each glucose molecule yields two G3P molecules.where kβ-cells, we do not differentiate the cytosolic and mitochondrial pyruvate pools in this model. For simplicity, we assume that mitochondrial pyruvate is decarboxylated only in a process catalyzed by PDH. The equation describing [Pyr] change over time isPyruvate is the main product of glycolysis. Due to lack of information on pyruvate concentrations at the sub-cellular level in mmit is the relative mitochondrial matrix fraction of the cell volume.where V+ flux due to pumping in the ETC. We assumed that 10 and 6 protons are pumped by each NADH and FADH2 oxidation, respectively m over timeWe assumed that the NADH production rate in mitochondria is determined by the reaction rate of pyruvate decarboxylation. The mitochondrial NADH concentration is decreased by the action of the ETC, during which NADH is converted to NADNADHm is the rate constant of NADH consumption in mitochondria. The concentration of pyridine nucleotides is assumed to be conserved in the mitochondrial matrix:where the stoichiometric coefficient 4.6 in the numerator arises from the number of NADH molecules that are produced from each pyruvate molecule. The coefficient 0.1 indicates that 10 protons are transported by the ETC for each NADH consumed . kNADHm tm is the total concentration of pyridine nucleotides in the mitochondrial matrix  Due to the rapid export of mitochondrial ATP to the cytosol via the ATP/ADP transporter no limitation for this process was suggested. (2) ADP binding is in equilibrium (i.e. the binding reactions are fast compared to the other processes included in the model of ATP dynamic).β-cells is mostly for ion transport, biosynthesis, and secretion. The rate of ATP utilization is a complex function of the concentrations of ATP, [Ca2+]c, glucose and numerous other factors, but almost nothing is known about the quantitative relation between them. Clearly, there is some basal level of ATP consumption in a cell at low glucose and [Ca2+]c concentrations. There is evidence that ATP hydrolyzing processes are accelerated during a glucose-induced increase in [Ca2+]c in β-cells, for example, ATP consumption can be increased due to increased Ca2+ pump activity in the plasma membrane and endoplasmic reticulum c change in cytoplasm to simulate the oscillations of the mitochondrial parameters in β-cells. The model described in this section is based on a simple model  gives the total flux in the mitochondria matrix per unit cell volume.A factor (0.31) was used to convert picomoles per islet from metabolism experiments to the cytoplasmic millimolar terms of a single  in Ref. ). The mia volume . In linem, [NADH]c, Ψm, [Ca2+]m, [ATP]c, Vp and [Ca2+]c. Coefficients are shown in Tables The general concentration of intramitochondrial and cytoplasmic adenine and purine nucleotides were kept constant during simulations Table . Model pc, Ψm, Ca+m, [ATP]Simulations were performed as noted previously for an idealized mean individual cell using the software environment from "Virtual Cell" . To http://www.nrcam.uchc.edu in "MathModel Database" on the "math workspace" in the library "Fridlyand" with the name "GlucoseSensitivity-1" for the general model and with name "GlucoseSensitivity-2" for the general model that also includes independent [Ca2+]c oscillations.This model is available for direct simulation on the website "Virtual Cell" ATP: ATP-sensitive K+ channels; LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase; PYR: pyruvate; ROS: reactive oxygen species; TCA: tricarboxylic acid; Tfam: mitochondrial transcription factor A; UCP2: uncoupling protein 2; Ψm: mitochondrial membrane potential; Subscript c: cytoplasmic compartment; m: mitochondrial compartment.ATP/ADP: ratio of ATP to ADP; ETC: electron transport chain; EtBr: ethidium bromide; GK: glucokinase; Glu: glucose; GSIS: glucose-stimulated insulin secretion; G3P: glyceraldehyde 3-phosphate; KThe authors declare that they have no competing interests.All authors  contributed equally to the formulation of the model, the estimation of parameters, the biological interpretations and conclusions, and the writing and editing of the manuscript. LF performed construction and simulation of the initial mathematical model. Both authors read and approved the final manuscript.


The Ca2+ responses in skeletal muscle, however, could not be explained by oxidative stress.Chemotherapy-induced fatigue is a multidimensional symptom. Oxidative stress has been proposed as a working mechanism for anthracycline-induced cardiotoxicity. In this study, doxorubicin (DOX) was tested on skeletal muscle function. Doxorubicin induced impaired  Fatigue, defined as sustained exhaustion and decreased functional capacity not relieved by rest, is one of the most common side effects of chemotherapy in cancer patients. Chemotherapy-induced fatigue has been associated with asthenia . This inex vivo as described earlier , electron chain inhibitors (rotenone 0.1–10 μM or piericidin A 0.25–2.5 μM) and antioxidants (N-acetyl cysteine (NAC) 10 μM–1 mM or trolox 10 μM–1 mM) was studied.Contractile characteristics of the extensor digitorum longus (EDL) from male CD2F1 mice, 7–9 weeks old, were assessed t=0 and t=120 min were identical for all parameters measured  and after a shorter incubation period (⩾0.5 h)   and 2B. (⩾0.5 h)  decrease  was performed. Stimulation after 2 h of incubation with DOX resulted in impaired maximal force, contraction and relaxation velocity  at the sin vitro model to examine if DOX affected Ca2+ fluxes. An overnight incubation with DOX resulted in increased calcium responses to ATP and caffeine  to release Ca2+ from the sarcoplasmic reticulum (SR). Caffeine can activate the RyR directly. If the purigenic receptor is stimulated by ATP, IP3 is formed, which in turn releases Ca2+ from the SR. Ca2+-induced Ca2+ release can then reinforce RyR stimulation. The elevated Ca2+ can be redistributed to mitochondria through the Ca2+ uniporter or to the SR through SERCA pump. Data from skeletal muscle SR fragments indicated that DOX could bind to Ryr-1 resulting in Ca2+ release from the SR  muscle, Caon phase . This efon phase  40 Hz. Ton phase . This mi calcium  and withgnancies . Patientgnancies . Therefo


Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify  rgs3 gene function produced body patterning defects like those observed with loss of wnt5b gene function. Analysis of endogenous calcium release dynamics in developing zebrafish revealed that both rgs3 and wnt5b are required for appropriate frequency and amplitude of calcium release. Our results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development.Vertebrate development requires communication among cells in order to define the body axis . Secreted factors such as Wnts play key roles in a vast array of cellular processes, including patterning of the body axis. One arm of the Wnt-signaling network, the non-canonical pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GAPs and are uniquely situated to control the amplitude of a Wnt signal. Here, we combine cellular, molecular, and genetic analyses with high resolution calcium imaging to identify a role for RGS modulation of Wnt-mediated calcium release dynamics and developmental patterning events. We find that loss of  Drosophila). β-catenin-independent Wnt signaling has also been shown to lead to calcium (Ca2+) release as well as activation of Rac, Rho and other cytoskeletal components in vertebrates 2+ signaling The Wnt signaling network is classified into β-catenin-dependent and β-catenin-independent pathways 2+ release dynamics during epiboly, gastrulation, convergent extension and organogenesis 2+ release events, aperiodic transient fluxes found mainly in the enveloping layer and dorsal forerunner cells 2+ levels in the deep cell layer and yolk syncytial layer 2+ transients are, in part, modulated by Wnt5 wnt5b genetic mutant (pipetail) shows reduced Ca2+ release hecate shows ectopic Ca2+ release 2+ release results in alteration of dorsal ventral patterning, cell movement and left-right patterning 2+ release, both transient and sustained, translate into distinct developmental outputs Zebrafish embryos demonstrate Ca2+ release in zebrafish embryos Wnts interact with receptors of the Frizzled (Fz) family G protein signaling is regulated by the lifetime of active Gα and βγ subunits. Activated Gα subunits have an intrinsic GTPase activity that converts the GTP-bound conformation to the Gα-GDP bound conformation allowing reassembly with Gβγ subunits to form the inactive Gαβγ heterotrimer rgs3, which was identified in an expression screen in zebrafish rgs3 is expressed in overlapping and adjacent domains with wnt5b at multiple stages of zebrafish development. Morpholino knockdown of rgs3 in zebrafish embryos causes convergence and extension (CE) defects that resemble phenotypes observed in the wnt5b genetic mutant, pipetailrgs3 and wnt5b. Additionally, we describe endogenous Ca2+ release dynamics during somite stages and show that Rgs3 and Wnt5b impact the frequency of Ca2+ release. Moreover, we show that Rgs3 modulates the extent and duration of Wnt5b induced Ca2+ activity. Functional analyses show that both the rescue of the rgs3 knockdown defect and impact on Wnt5-induced Ca2+ release requires a key asparagine in the RGS domain of Rgs3 necessary for Gα binding and acceleration of its GTPase activity. This research identifies a link between Wnt5b signaling and Rgs3 activity relative to the frequency of Ca2+ release, thus revealing obligatory roles for RGS proteins in vertebrate development in the context of the whole animal. Our results also demonstrate that the biological outcome of Wnt signaling depends greatly upon regulating the duration of its signal, as shown here with Rgs3.To investigate potential roles of RGS proteins in vertebrate development, we utilize gene knockdown in zebrafish. We focus on rgs3 was identified in an expression screen during early somitogenesis stages rgs3 expression begins during the blastula period shortly after zygotic transcription initiates , and persists through the segmentation period (10–24hpf) (In Situ Hybridization (WMISH) demonstrated ubiquitous rgs3 expression during epiboly and gastrulation stages. During somite stages (10–20 hpf), rgs3 expression resolves in the somites, tailbud, and brain (rgs3 expression in the midbrain/hindbrain boundary as demonstrated by overlap with the molecular marker engrailed 1 (eng1) at 28 hpf  portion of developing somites . As Wnt5b is a secreted ligand, the proximity of rgs3 to wnt5b producing cells suggests that Rgs3 may function in modulating Wnt5b signaling.Zebrafish 0–24hpf) . Whole Mnd brain , with dit 28 hpf , and enr somites . rgs3 an tailbud . rgs3 ex vesicle , a ciliawnt5b induces increased Ca2+ release during the blastula stage in a G protein dependent manner rgs3 over-expression is sufficient to negatively regulate Wnt5b signaling . A ratio image is derived as the quotient of the 340-nm image divided by the 380-nm image on a pixel-by-pixel basis, and provides spatial and temporal measurement of Ca2+ dynamics. Ca2+ release activity was monitored over a 75 minute time course during the blastula stage. Sequential ratiometric images were analyzed by a subtractive algorithm to identify changes in Ca2+ release activity (transients) over time as well as the location of the activity as described previously 2+ release mapped on the embryo. The number of Ca2+ transients during the cellular blastoderm stage is represented by height of the peaks and color coded where purple is low and yellow/red is high number of events. The composite image of a wild-type (wt) embryo during the blastula stage indicates endogenous Ca2+ levels throughout the embryo (2+ release in an embryo injected with wnt5b (rgs3 with dextran-conjugated Texas Red (TxR) lineage tracer into a subset of cells in embryos uniformly expressing wnt5b co-mixed with Fura-2 supports that rgs3 is sufficient to suppress wnt5b induced Ca2+ release as demonstrated by the reduction of Ca2+ levels . We eval release  as demon embryos . To rulegh 24hpf . Immunosrgs3 during development. To knockdown Rgs3, we utilized antisense morpholino oligonucleotides (MO) rgs3 5′UTR (MO and MOb) or splice junction (SA) (rgs3 MO-injected embryos have shorter A-P extension and a kinked tail (wnt5b (pipetail) genetic mutant rgs3 morphants display tighter packed and rounded somites  axis leading to closer spaced myoD  . All MOsion (SA) . In contked tail  reminisc somites . To eval midline . In cont somites . Additio embryos . rgs3 moA-P axis . Togethergs3 MO which induced morphological somite defects . To demonstrate a transient, a region of interest (ROI) is noted by dashed circle   . A simil (n = 3) , and S2K2+ release dynamics during somitogenesis, we next determined the impact of rgs3 knockdown. From the development of somite 6 to somite 12, rgs3 morphants have statistically more Ca2+ transients, with an average of 21.7 per hour (n = 3), when compared to wt embryos  genetic mutants wnt5b morphant embryos show a statistically reduced number of Ca2+ transients, averaging 1.3 per hour (n = 2) during the segmentation period   . The redDrosophila genetics places active G protein signaling upstream of Dvl wnt5b co-expression reduced Dvl-MT levels  results in a mild phenotype (rgs3 MOsa (0.8 ng), which produced a phenotype (rgs3 MOsa or wnt5b MO did not induce any severe defects (wnt5b MO (2 ng) combined with rgs3MOsa (0.8 ng) resulted in a 92% penetrance of severe defects , partial knockdown of rgs3 could lead to discordant changes in the frequency and amplitude of Ca2+ release result in the dramatic phenotypic penetrance and severity.Studies have shown that increased Wnt/Fz signaling leads to degradation of Dvl T levels . Reductirphology  and moleand myoD . Compareand myoD , low doshenotype . We nexthenotype  indistinhenotype . Individ defects . However defects . Our Ca22+ release dynamics during somitogenesis. The frequency of Ca2+ transients as well as the observation of sustained Ca2+activity in the trunk and tail region are consistent with previous reports of Ca2+ activity during zebrafish somitogenesis 2+ release upon rgs3 knockdown.These results provide new evidence for an essential role of Rgs3 in modulating the duration of Wnt5b signaling. We show that Rgs3 is necessary for proper gastrulation and somite patterning during zebrafish development. These actions of Rgs3 require its ability to interact with and accelerate the rate of GTP hydrolysis by G proteins, as revealed by studies employing an Rgs3 mutant defective in these activities. We describe endogenous Ca2+ signaling was first demonstrated in 1997 wnt5b induced Ca2+ release and this ability requires GAP activity consistent with the known role of G proteins in the activation of Wnt signal transduction pathways 2+ release that this dramatic impact on Ca2+ dynamics in the somites is dependent upon Wnt5 supporting that Wnt/Ca2+ signaling activity is an in vivo target of RGS proteins. Moreover, rgs3 demonstrates a complex genetic interaction with wnt5b. rgs3 is expressed in and near wnt5b expressing tissues and we find that combined low doses of wnt5b MO and rgs3 MOsa result in a large penetrance of severe somite defects which is not observed during their individual knockdown. Our data suggest that both the frequency and amplitude of wnt5b signaling must be tightly regulated to result in correct cell movement and somite patterning.RGS proteins were identified as negative regulators of G protein signaling in the mid 1990s 2+ release activity in small populations of cells, as well as, sustained activity in a large region of cells pipetail genetic mutants 2+ activity at the somite boundaries (data not shown). It is of interest to determine if interactions between rgs3 and wnt5b influence directed vascular outgrowth.Wnt5b modulates both transient Cain vivo physiological analysis in whole embryos has provided compelling functional insight into the developmental role of RGS proteins in the Wnt signaling network.Modulation of G protein signaling (impacting frequency as well as duration) is likely to influence directed cell migration, vascular development as well as numerous other developmental processes Adults were maintained in a 14-hour light / 10-hour dark cycle at 28°C. Embryos were collected from natural pairwise matings and staged by hours post fertilization (hpf) at 28.5°C and morphological criteria described in Kimmel et al. rgs3 (clone IBD5096) was isolated in an expression screen in zebrafish rgs3 was generated by RT-PCR and directionally cloned (5′-3′) into the pCS2+, pCS2+ myc or pCS2+ Flag expression vector. The Quick Change II site-directed mutagenesis kit (Stratagene) was used to generate an Asparagine (N) to Alanine (A) substitution at amino acid 109 which is located in the RGS domain of Rgs3. Synthetic RNA was then in vitro transcribed using SP6 RNA polymerase and the mMessage mMachine system (Ambion).rgs3 MO and rgs3 MOb) to inhibit translation and an intron-exon junction in the RGS domain (rgs3 MOsa) to alter splicing. As a control rgs3 MOmm (5 bp mismatch in lowercase letters) was designed (Gene-Tools):Antisense morpholino oligonucleotides (MO) were designed to target the 5′-UTR/ATG  was co-injected with 20 ng rgs3 MO. Injected embryos were characterized by morphological and molecular analysis.MOs (5–20ng) were pressure-injected into one cell-stage embryos. For low-dose knockdown, 0.8ng Embryos were fixed overnight in 4% paraformaldehyde and dechorionated. Single label WMISH was performed as previously described myc-rgs3 or (N109A)myc-rgs3 (750 pg). To investigate Rgs3's impact on Dvl, C-terminal myc tagged zebrafish dvl2 (300 pg) was injected alone, with rgs3 MOsa (5ng), with wnt5b (250pg), and with both rgs3 MOsa (5ng) and wnt5b (250pg). Injected Embryos were allowed to develop to the appropriate stage (5 hpf and 24 hpf) before incubating in lysis buffer  at room temperature for 3 minutes. Embryos were then disrupted using a pestle, centrifuged at 13,000 rpm for 10 minutes at 4°C and the clear supernatant containing whole zebrafish protein was collected. Approximately 10µg of protein was loaded in each well and separated with SDS-PAGE gel electrophoresis. Proteins were transferred onto nitrocellulose membrane (Li-Cor) and incubated with the following primary antibodies: mouse anti-myc  and rabbit anti-β actin , and then incubated with the following secondary antibodies: IRDye800 anti-mouse  and IRDye680 anti-rabbit . The signal was visualized using the Odyssey Infrared Imaging System (Li-Cor).To compare levels of MT-Rgs3 and mutant MT-Rgs3, embryos were injected with either myc-rgs3 or (N109A)myc-rgs3 (200 pg) were fixed 1–3 hrs in 4% PFA/1× PBS at sphere/dome stage. Mouse anti–myc antibody , followed by goat-anti-mouse Alexa488 conjugated secondary antibody  was used to detect the rgs3 products. Nuclei were identified with 5 µM TO-PRO-3 (Molecular Probes). Embryos were mounted in an animal pole orientation in bridged coverslips and optically sectioned using two-channel imaging on a scanning laser confocal microscope, Leica TCS SP2. Wide-field fluorescence and bright–field images from a Zeiss Stemi M13 Bio Stereoscope were photographed using Axiovision (Zeiss) software and an Axiocam 5000 camera. Images were merged using Adobe Photoshop CS.Embryos injected with either 2+-sensing dye Fura-2 or Bis-Fura-2 (Molecular Probes) was injected into 1-cell zebrafish embryos. The excitation spectra are different between Ca2+ bound Fura-2 (340-nm) and Ca2+ free (380-nm) forms. By taking the ratio of the fluorescence intensity at these wavelengths an estimate of intracellular-free Ca2+ can be derived. To stimulate Wnt signaling, in vitro transcribed wnt5b RNA (400 pg) was co-injected with Fura-2 at the one cell stage. rgs3 or rgs3N109A RNA (400 pg) was unilaterally injected at the 16-cell stage mixed with dextran-conjugated Texas Red (TxR) lineage tracer. TxR distribution was determined by collecting a reference exposure at 540-nm excitation. For cellular blastoderm stage imaging, embryos were oriented in a lateral position in a glass-bottomed dish on a Zeiss axiovert epifluorescence microscope. Image pairs at 340 and 380-nm excitation wavelengths (510-nm emission) were collected at 15-second intervals. Each imaging session collected 300 image pairs. The ratio image, a pixel by pixel match of both excitation wavelengths, is calculated by computer software . The sequence of ratio images was processed and the Ca2+ fluxes (transients) were determined by a subtractive analog patterned after 2+-saturated and 380nm, Ca2+-free) imported for publication is encoded in 8 bits and converted to pseudocolor with low ratio (low Ca2+) represented by blue and high ratio (high Ca2+) represented by yellow/red.The ratiometric Ca2+ transients. Somite stage Ca2+ transients were defined as a localized increase in Ca2+ which persists no longer than thirty seconds.For somite imaging, 2–6 somite stage embryos were oriented in low melt agarose (0.4%) in a dorsal or lateral orientation. Time courses collected images pairs until 12–15 somite stage at 15-second intervals (Approximately 1000 images pairs). Image pairs were converted to ratio images as described above. Sequential ratio images were manually analyzed for changes in Ca2+ transients in morphant embryos were made using One-Way Analysis of Variance (one-way ANOVA) with Tukey HSD test p-values reported.Calculations for MO rescue experiments were made using the Fisher's exact test and the two-tailed p-value was reported. Calculations for analysis of somite stage CaFigure S1rgs3 expression is adjacent and overlapping with wnt5b, related to rgs3 compared to wnt5b in zebrafish development. Whole Mount In Situ Hybridization was utilized to compare the spatial expression of wnt5b to rgs3. WMISH of 14hpf (A–D) and 24hpf (E–F) Wt embryos. Lateral (A–C and E) and dorsal (D and F) views illustrate that rgs3 is expressed in the developing somites and posterior tail (A–E). Co localization of wnt5b and rgs3, determined by double label WMISH with wnt5b (red) and rgs3 (blue), shows adjacent and overlapping expression domains around Kupffer's vesicle (C) and in the tailbud (D). Double label WMISH with rgs3 (blue) and engrailed1 (red) highlight that rgs3 is expressed in the midbrain/hindbrain boundary (E). Sense probes (negative control) gave no specific hybridization signal.(1.19 MB TIF)Click here for additional data file.Figure S2rgs3 impacts segmentation stage calcium dynamics, related to 2+ as blue and high Ca2+ as yellow/red (B–J). Representative ratio images of 6 somite stage , 8 somite stage  and 10 somite stage  embryos. Arrowheads indicate large Ca2+ transients in rgs3 morphant embryos (E–G) that are not observed in Wt (B–D) or wnt5b morphant embryos (H–J). The number of Ca2+ transients per hour observed in embryos oriented in a lateral posterior view from 6 to 12 somite stage is represented function of developmental age is represented graphically (K).(2.07 MB TIF)Click here for additional data file.Table S1rgs3 knockdown, related to Rescue efficiency of (0.04 MB DOC)Click here for additional data file.Video S12+ dynamics during zebrafish somitogenesis, related to 2+ represented by blue and high Ca2+ represented by yellow/red.Wt, Ca(2.58 MB AVI)Click here for additional data file.Video S22+ dynamics during zebrafish somitogenesis, related to rgs3 morphant zebrafish embryo oriented in a dorsal posterior view) consisting of pseudocolored ratio images derived from image pairs (340 and 380-nm excitation wavelengths) collected in 15-second intervals over a two hour period with low Ca2+ represented by blue and high Ca2+ represented by yellow/red.rgs3 morphant, Ca(2.58 MB AVI)Click here for additional data file.Video S3rgs3 RNA, Ca2+ dynamics during zebrafish somitogenesis, related to rgs3 RNA zebrafish embryo oriented in a dorsal posterior view) consisting of pseudocolored ratio images derived from image pairs (340 and 380-nm excitation wavelengths) collected in 15-second intervals over a two hour period with low Ca2+ represented by blue and high Ca2+ represented by yellow/red.rgs3 morphant rescued with (1.23 MB AVI)Click here for additional data file.Video S42+ dynamics during zebrafish somitogenesis, related to 2+ represented by blue and high Ca2+ represented by yellow/red. Double label WMISH with rgs3 (blue) and engrailed1 (red) highlight that rgs3 is expressed in the midbrain/hindbrain boundary (E).wnt5b morphant, Ca(2.58 MB AVI)Click here for additional data file.


Differentiation inhibitory factor (nm23 protein) inhibited the induction of the differentiation of various leukaemic cell lines. We previously reported that nm23 genes (H1 and H2) were overexpressed in acute myelogenous leukaemia (AML) and nm23-H1 expression predicted the prognosis of AML, especially AML-M5. To clarify the correlation between French-American-British (FAB) classification and nm23 expression level and to clarify the involvement of nm23-H2 and nm23-H1 in patient survival, we investigated the relative levels of nm23-H1 and -H2 mRNA in 76 AML samples using the reverse transcriptase-polymerase chain reaction. We confirmed that the expression of both nm23-H1 and -H2 genes in AML samples from three different hospitals was significantly higher than that in normal blood cells (P < 0.0005). Overexpression of nm23-H1 was observed in each FAB AML-M1, -M2, -M3, -M4 or -M5 subtype, and the predictive effect of nm23-H1 expression on AML prognosis was shown in FAB AML-M2 and -M5 cases. Although overexpression of nm23-H2 was also found in each FAB subtype, the expression of nm23-H2 in AML-M1 and -M3 cells was not significantly higher than that in normal cells. Among AML subtypes, AML-M3 showed the lowest expression levels of both nm23 genes. To understand the relationship between nm23-H1 and -H2 expression levels, nm23 expression levels for all the AML cases were plotted and divided into four groups . A statistically significant correlation between the levels of expression of nm23-H1 and -H2 was observed (r= 0.726). Most AML-M3 cases belonged to group B, but not other types of AML. Analysis of survival probability between the groups showed that group B survived for significantly longer compared with group A. Furthermore, AML-M3 cases survived for significantly longer compared with non-M3 cases in the same group B. These data suggest that low expression levels of both nm23-H1 and -H2 are associated with good prognosis in AML patients.


Field-stimulated, whole-cell [Ca2+]i transients in hESC-CMs required Ca2+ entry through L-type Ca2+ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca2+ or treatment with diltiazem, an L-type channel inhibitor. Ca2+ release from the SR also contributes to the [Ca2+]i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca2+ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca2+ currents (ICa) and corresponding whole-cell [Ca2+]i transients in hESC-CMs and hFVMs, and the amplitude of both ICa and the [Ca2+]i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca2+]i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca2+ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca2+ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca2+ release by the ICa during EC coupling develops early in human cardiomyocytes.We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca These cells express expected cardiac markers and exhibit spontaneous action potentials (APs), [Ca2+]i transients, and contractile activity. At present, however, the mechanisms underlying excitation-contraction (EC) coupling in hESC-CMs are incompletely understood. Addressing this issue is critical for two fundamental reasons. First, hESC-CMs represent a unique model system in which to study the development of EC coupling in early human myocardium. Second, because of their tremendous expandability and unquestioned cardiac potential, hESC-CMs have considerable promise for eventual application in cell-based cardiac repair  can be induced to differentiate 2+ channels during the AP allow a small amount of Ca2+ to enter the cardiac cytoplasm. This Ca2+ influx causes a local [Ca2+]i increase that rapidly (<1 ms) activates nearby sarcoplasmic reticulum (SR) Ca2+ release channels  by the mechanism of Ca2+-induced Ca2+ release (CICR) 2+ stored in the lumen of the SR to flow into the cardiomyocyte cytoplasm causing a local increase in [Ca2+]i. These local Ca2+ release events, termed ‘Ca2+ sparks’, are considered the elementary Ca2+ release events of EC coupling2+ sparks can occur spontaneously, or they can be activated by the L-type Ca2+ current (ICa) ICa synchronizes the activation of multiple Ca2+ sparks, which sum to produce a large, whole-cell [Ca2+]i transient. As ICa inactivates, the probability of activation of Ca2+ sparks diminishes, thereby allowing the SR Ca2+ ATPase and Na+-Ca2+ exchanger to return the [Ca2+]i to resting levels 2+]i is graded by the amplitude of ICaUnlike in hESC-CMs, the mechanisms underlying EC coupling in mammalian adult ventricular myocytes have been the subject of intense investigation for many years and so are comparatively well-understood .In this study, we examined the mechanisms of EC coupling in hESC-CMs, as well as in ∼100 day old human fetal ventricular myocytes (hFVMs), which serve as a useful comparison cell type of known age. Using a variety of techniques including fluorescent CaFor all experiments, H7 hESCs et al.2+-free Tyrode's solution  for 5 minutes, followed by a switch to a solution also containing 1% collagenase I , 0.01% protease, and 0.08 mM CaCl2. After approximately 10 minutes of digestion, the left ventricular tissue was removed, minced and filtered through nylon mesh. The resultant dissociated ventricular myocytes were maintained in Dulbecco's MEM at 25°C for up to 4 hours before use.Human fetal hearts  were obtained from the University of Washington Birth Defects Research Laboratory under a waiver from the University's Institutional Review Board (IRB) for Human Subjects. The IRB determined that this work, which involved anonymous human biological materials received from this depository, is not considered human subjects research (IRB Determination # 08-0062-N). The NIH-funded Birth Defects Research Laboratory tissue distribution program has been separately approved by the IRB  and operates in fully compliance with all relevant state and federal laws and regulations. All donors provide written informed consent prior to donating tissues to this depository, and all donated tissues would otherwise be discarded. Ventricular myocytes were then dissociated from these fetal hearts using enzymatic methods modified from those described by Ufret-Vincenty 2+]i using the fluorescent Ca2+ indicator Fluo-4 . For experiments that involved the simultaneous measurement of electrophysiological signals and [Ca2+]i, cells were loaded with the penta-potassium salt of Fluo-4 (50 µM) through the patch pipette. For measurements of [Ca2+]i that did not involved patch-clamping , myocytes were loaded with the membrane permeable acetoxymethyl-ester form of Fluo-4 (Fluo-4 AM) as previously described We measured changes in [Ca2+]i and Ca2+ sparks was performed using a BioRad Radiance 2000 or Nikon Swept Field confocal system  coupled to a Nikon TE300 inverted microscope equipped with a Nikon 60× oil immersion lens (NA = 1.4). Images were analyzed with custom software written in IDL language . Ca2+ sparks were identified using a computer algorithm similar to the one described by Cheng et al.2+]i transient evoked by the application of a Ca2+- and Na+-free (substituted with N-methyl-D-glucamine) solution containing 20 mM caffeine  was used as an indicator of SR Ca2+ content 2+ load, cells were subjected to a minimum of 10 preconditioning pulses (1 Hz) before caffeine was applied. Calibration of fluorescence signals was performed using the ‘pseudo-ratio’ equation Confocal imaging of whole-cell i transient during EC coupling in hESC-CMs. APs were evoked via field stimulation (1 Hz). [Ca2+]i was recorded in cells loaded with the fluorescent Ca2+ indicator fluo-4 using confocal microscopy (see 2+), APs evoked large, cell-wide [Ca2+]i transients in hESC-CMs (2+]i was 4.6±0.4 F/F0 (n = 19 cells). [Ca2+]i rose in hESC-CMs after activation of the AP: the time-to-peak of the evoked [Ca2+]i transient was 150±25 ms. Analysis of the decaying phase of the [Ca2+]i transient revealed the time it took to decay to 50% amplitude of its amplitude (T50) was 245±45 ms. Note that the time-to-peak and T50 of the [Ca2+]i transients in hESC-CMs are similar to those reported for [Ca2+]i transients in adult human ventricular myocytes 2+ eliminated AP-evoked [Ca2+]i transients in hESC-CMs, demonstrating a requirement for Ca2+ influx during EC coupling in these cells . Consistent with our hypothesis, diltiazem (10 µM) eliminated the [Ca2+]i transient in these cells  inhibitor thapsigargin (1 µM) . We next repeated this experiment using the reversible SERCA inhibitor cyclopiazonic acid . 2+]i transients from a representative hESC-CM under control conditions, during CPA treatment, and after the SERCA pump inhibitor was washed out. CPA decreased [Ca2+]i transient amplitude to 30±10% , but this effect was partially reversed following drug washout . Taken collectively with the data in 2+ release amplifies the Ca2+ influx via L-type Ca2+ channels and is a major contributor of Ca2+ during the evoked global [Ca2+]i transient during EC coupling.We examined the role of SR Can (1 µM) . Thapsig2+ influx is by itself sufficient to produce the [Ca2+]i transients observed in hESC-CMs (i.e. model 1 discussed in the Introduction), we investigated whether L-type Ca2+ channels activate SR Ca2+ release during EC coupling through a loose (i.e. model 2 above) or a tight, local control mechanism (i.e. model 4 above). If SR Ca2+ release in hESC-CMs is activated by local Ca2+ signals produced by closely apposed L-type Ca2+ channels, one would expect the amplitude of the [Ca2+]i to be a finely graded function of the amplitude of ICa. On the other hand, if SR Ca2+ release in hESC-CMs is activated by Ca2+ signals of variable strength (as would be expected with variations in the relative location of RyRs and L-type Ca2+ channels) or by spontaneous SR Ca2+ release alone (i.e. model 3 above), there would be a poor correlation between the amplitude of ICa and the associated [Ca2+]i transient in these cells. To determine which of these potential scenarios apply to hESC-CMs, we simultaneously recorded ICa and [Ca2+]i in hESC-CMs during step depolarizations (200 ms duration) from the holding potential of −50 mV to test potentials ranging from −40 to +60 mV  and presumably more advanced developmental stage.Having ruled out the possibility that Cao +60 mV . We alsoICa and global [Ca2+]i transients in hESC-CMs and hFVMs. The amplitude of ICa and the associated [Ca2+]i transient varied with the magnitude of the test membrane potential in both cell types  and 11.6±1.6  pA/pF in hESC-CMs and hFVMs, respectively. The amplitude of the [Ca2+]i transient at the same potential was 1097±97 and 902±77 nM (p = 0.15) in hESC-CMs and hFVMs, respectively.Membrane depolarization evoked large ll types . InteresICa  is that this gain factor will be high at negative potentials  but will decrease with depolarization to increasingly positive test potentials . This prediction holds true in mammalian adult ventricular myocytes Moreover, as previously reported in adult ventricular myocytes ICa  and the ansients  were bel2+ (∼+120 mV), L-type Ca2+ channels open, but the Ca2+ influx through these channels is low due to a weak driving force. However, upon repolarization from such extremely positive test potentials (e.g. repolarization from +100 mV to −70 mV), there is a rapid increase in driving force, and a “tail” ICa is evoked. Because L-type Ca2+ channels deactivate rapidly (τ of deactivation<1 ms), these tail ICa currents are short-lived. In adult ventricular myocytes, tail ICa currents can also induce [Ca2+]i transients, a phenomenon that has been attributed to CICR initiated by the Ca2+ influx during repolarization 2+]i transients is considered strong evidence for the local control hypothesis, as the extremely brief tail ICa is thought to allow only a small amount of Ca2+ influx and so is only able to activate RyRs within a tight, local domain. The presence of tail [Ca2+]i transients also distinguishes cardiac-type EC coupling from that in skeletal muscle, which operates on a charge-coupled mechanism and so repolarization does not elicit SR Ca2+ release 2+]i transients in response to repolarization to −70 mV following a voltage step to +100 mV  were largely eliminated by treatment with ryanodine (10 µM), an effect that indicates a significant contribution by release from RyR-gated SR Ca2+ stores (2+]i transients (evoked by repolarization from +100 mV to −70 mV) from a baseline of 945±37 nM to 250±10 nM, and it reduced the amplitude of [Ca2+] transients (evoked by a depolarizing step to 0 mV) from 853±52 nM to 269±8 nM (n = 6 cells per condition).When adult ventricular are depolarized to highly positive test potentials close to the Nernst equilibrium potential for Ca +100 mV . Of note+ stores . In part2+ release from the SR is graded by the amplitude of ICa, likely via local Ca2+ signaling between closely apposed L-type Ca2+ channels and RyRs in hESC-CMs (i.e. model 4).Collectively, these data indicate that Ca2+ channels (Cav1.2) and ryanodine receptors (RyRs) are co-localized within specific domains near the sarcolemma. To test this hypothesis in hESC-CMs, we examined the subcellular distribution of type 2 RyRs (RyR2s) and L-type Ca2+ channels in hESC-CMs using confocal immunofluorescence approaches. As illustrated by 2+ channels throughout the sarcolemma of hESC-CMs. Interestingly, L-type Ca2+ channels (red) and RyR2s (green) were co-localized within small clusters near the surface of the cell (2+ channels and RyR2 clusters was 1.2±0.1 µm (n = 55) and 1.0±0.1 µm (n = 41), respectively. We performed quantitative co-localization analysis of L-type Ca2+ and RyR2-associated fluorescence on a pixel-by-pixel basis. The statistical significance of the co-localization of these two proteins is supported by Mander's overlap and Pearson correlation values of +0.88 and +0.83, respectively. These data indicate that L-type Ca2+ channels are co-localized with RyR2s within small sarcolemmal clusters in hESC-CMs.Another testable prediction of the local control model of EC coupling is that L-type Cathe cell . The dia2+ release from the SR of hESC-CMs and hFVMs, we asked whether these cell types exhibit Ca2+ sparks, the elementary release events during EC-coupling. In fact, spontaneous Ca2+ sparks were routinely observed in both hESC-CMs and hFVMs. Representative sparks and associated parameters from both cell types are depicted in 2+ sparks in these cells is presented in 50 were similar in both cell types . Interestingly, however, the Ca2+ spark rate (defined as the number of Ca2+ sparks per sec per cell) was nearly four-fold higher in hFVMs (n = 7) than in hESC-CMs . Ca2+ spark amplitude and time-to-peak were also greater in hFVMs (n = 86 events) than in hESC-CMs .To better understand the mechanisms underlying Ca2+ load modulates Ca2+ spark activity 2+]i transient as an indicator of SR Ca2+ load  was applied after conditioning each cell with a train of ten APs. We found that the amplitude of the caffeine-induced [Ca2+]i transient was higher in hFVMs  than in hESC-CMs , suggesting that steady-state SR Ca2+ load is greater in hFVMs than in hESC-CMs.Finally, because SR Caa2+ load . To ensu2+ release during the AP occurs at special structures named Ca2+ release units (CRUs) in which L-type Ca2+ channels are in close proximity (about 10–12 nm) to RyRs in the junctional SR 2+ channels in these regions of the cell allow enough Ca2+ influx to produce a local, junctional increase in [Ca2+]i (10–100 µM) to activate nearby RyRs via CICR, resulting in Ca2+ sparks 2+ and [Ca2+]i does not reach high enough levels in neighboring sites to induce their activation. This prevents the regenerative, all-or-none [Ca2+]i transient that would be predicted by a global control mechanism. Instead, the amplitude of the [Ca2+]i transient is produced by the temporal and spatial summation of Ca2+ sparks activated by ICa during the AP. The amplitude of the [Ca2+]i transient in adult ventricular myocytes is graded by the amplitude of ICa, which determines the probability of Ca2+ spark recruitment during an AP.In adult ventricular myocytes, activation of SR Ca2+]i transients and express key Ca2+ handling proteins, including SERCA2b, the Na+/Ca2+ exchanger, and RyR2, there has been considerable controversy as to the fundamental mechanisms of excitation-contraction coupling in these cells et al2+]i transient rise observed in hESC-CMs results from trans-sarcolemmal entry via calcium channels and that these cells have little or no release from internal Ca2+stores. By contrast, Satin et al2+stores, even at early stages (e.g. as soon as 2 days following the appearance of spontaneous beating activity). A third group, Liu et al. 2+stores in hESC-CMs, albeit only in a minority of cardiomyocytes. Of note, all three groups largely relied on hESC-CMs generated using the “historical” method of embryoid body differentiation ). The present study is the first to examine intracellular Ca2+ signaling in hESC-CMs resulting from our recently reported guided differentiation protocol . This method results in preparations of substantially higher cardiac purity , greatly facilitating biophysical studies with hESC-CMs, but it could in principle result in cardiomyocytes with a different functional phenotype. For example, FCS is known to retard cardiac differentiation Although hESC-CMs are known to exhibit [Ca2+ sparks and exhibit robust ICa and [Ca2+]i transients. ICa density in hESC-CM is similar to that of hFVM as well as adult human, mouse, and rat ventricular myocytes ICa density is established early in the differentiation of ventricular cardiomyocytes and that, during development, myocyte hypertrophy is accompanied by a concomitant increase in L-type Ca2+ channel expression (presumably Cav1.2) that leads to a relatively stable ICa density throughout development.Here, we report that that hESC-CMs produce CaICa and [Ca2+]i transients in hESC-CMs, are consistent with a model in which activation of SR Ca2+ release is tightly regulated by Ca2+ signals via L-type Ca2+ channels during EC coupling. The data supporting this model are compelling. First, Ca2+ influx via functional L-type Ca2+ channels is necessary for EC coupling in hESC-CMs. Second, Ca2+ influx via L-type Ca2+ channels activate SR Ca2+ release in these cells, thereby producing a global increase in [Ca2+]i that triggers contraction. Third, SR Ca2+ release is not all-or-none in hESC-CMs. Instead, as in adult ventricular myocytes 2+ release is finely graded by the amplitude of the L-type Ca2+ current during EC coupling. Fourth, large [Ca2+]i transients were evoked by tail ICa during repolarization from +100 mV to −70 mV. Fifth, EC coupling gain is larger at negative potentials where ICa is small (i.e. −40 mV), but the driving force for Ca2+ influx is large, than at more positive potentials in which ICa is maximal (i.e. 0 mV), but the driving force for Ca2+ entry is lower. Sixth, RyR2 and L-type Ca2+ channels co-localize in specific regions of the sarcolemma of hESC-CMs. Collectively, these findings strongly indicate that the establishment of tight, local control of EC coupling is an early event in the development of human cardiomyocytes. Note that this conclusion distinguishes hESC-CMs from developing cardiomyocytes in model species, including mice The findings in our study, which is the first to directly examine the relationship between ICa, we found that the amplitude of the evoked [Ca2+]i transient in hESC-CMs was similar to that of hFVMs as well as adult human 2+ influx and SR Ca2+ release  translates into a global [Ca2+]i transient during the physiological AP in hESC-CMs. Interestingly, the relative contribution of ICa (≈20%) and the SR (≈80%) to the [Ca2+]i transient in these cells is similar to that of adult human ventricular myocytes 2+ influx and release from intracellular release are established early in cardiac biogenesis and remain stable throughout development. Multiple factors come into play in the generation of the [Ca2+]i transient in ventricular myocytes, including the amount of Ca2+ influx, SR Ca2+ release, the buffering capacity of the cell as well as the rate and capacity of Ca2+ extrusion, and SR Ca2+ re-sequestration mechanisms. For example, while the absolute values for cytosolic buffering capacity, accessible volume, Ca2+ influx and SR Ca2+ release likely differ between hESC-CMs, hFVMs, and adult ventricular myocytes, it is clear their relative proportions lead to comparable AP-evoked changes in [Ca2+]i in these cells.As with the magnitude and kinetics of the 2+ sparks, and their sparks were similar in amplitude, duration, and spatial spread to those reported in adult cardiomyocytes ICa appears fairly early in the development of ESC-CMs 2+ sparks. We infer that this early development of CRUs is also likely to be true in human heart development in situ. Ca2+ spark rate was lower in hESC-CMs than in hFVMs presumably because SR Ca2+ load was higher in hESC-CMs than in hFVMs these cells 2+ load than hESC-CMs, but Ca2+ sparks and whole-cell [Ca2+]i of similar amplitude? Our data do not provide an answer to this difficult question. One intriguing possibility is that the number of RyRs underlying a Ca2+ spark differs between hESC-CMs and hFVMs. Thus, while the total Ca2+ flux through each RyR channel may be smaller (due to lower SR Ca2+ load) in hESC-CMs than in hFVMs, a larger number of these channels per CRU in hESC-CMs results in Ca2+ sparks of similar amplitude in both cell types. If correct, this implies that, while hESC-CMs have functional CRUs capable of producing Ca2+ sparks, the development of these units is likely incomplete. Future studies should examine the mechanisms underlying the development of CRUs and Ca2+ sparks in hESC-CMs in more detail.hESC-CMs and hFVMs also showed CaThe Kamp ICa and [Ca2+]i in hESC-CMs. Furthermore, our findings support the view that tight, presumably local, control of EC coupling is an early event in human cardiac development. Note that this conclusion distinguishes hESC-CMs from developing cardiomyocytes in model species, including mice 2+ channels and RyRs—not their density or coupling strength—that changes during the maturation of human cardiomyocytes.To conclude, our data clearly indicate that there is a close relationship between


Mn2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca2+ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca Light-evoked cone signals are regulated by two separate gain control mechanisms: the phototransduction pathway at the input in the outer segment (OS) and the release rate of synaptic vesicles at the output in the synaptic terminal. Both pathways are dynamically regulated by changes in intracellular calcium concentration [Ca2+ influx through L-type voltage gated channels stimulates neurotransmitter release in cones i was suggested to extend the operating range of the cone output into the direction that is hyperpolarized vis à vis the L-type channel threshold Ca2+]i baseline in hyperpolarized cones, potentially offsetting toxic effects of powerful Ca2+ clearance mechanisms 2+ - permeable channels is facilitated by hyperpolarization, potentiated by depletion of intracellular stores and is characterized by pharmacology that shares many features with store-operated Ca2+ entry (SOCE) that has been extensively studied in non-excitable cells We report a novel pathway in cone inner segments that dominates steady-state [Ca2+ concentration in isolated salamander cones was measured by analyzing [Ca2+]i signals from cells loaded with the high affinity indicator dye Fura-2. This non-invasive approach ensured that important cytosolic molecules potentially involved in modulation of Ca2+ fluxes were not lost or compromised. Salamander retina is advantageous for imaging studies because of the large size of salamander cone inner segments and synaptic terminals (5–10 µm diameter) in situ calibration of cytosolic [Ca2+]i within all classes of salamander cone Intracellular Ca2+ channels therefore [Ca2+]i tends towards a “resting” value associated with the saturating response to light. In dissociated light-adapted cones, the average resting [Ca2+]i value in the cell bodies was low, at 50±3 nM. Removal of Ca2+ from extracellular saline caused a further [Ca2+]i decline to 13±1 nM  with time constants of decay that were fit by a single exponential of τ = 2.0±0.3 min . Perfusion with control 2 mM Ca2+-containing saline led to [Ca2+]i levels similar to control values  of Gd3+ and La3+ reduced [Ca2+]i baseline to 24±4 nM  and 24±1 nM , respectively. This suggests that tonic Ca2+ entry observed in hyperpolarized cones occurs via Ca2+ channels located in the plasma membrane.Baseline Ca2+ influx pathway in the cone IS 2+]i. 0 [Ca2+]o-induced decline in [Ca2+]i was not mimicked by Cd2+, a universal inhibitor of voltage-operated Ca2+ channels. At 100 µM, Cd2+ had no effect on [Ca2+]i (n = 16). Consistent with this observation, two antagonists of L-type channels, D-cis-diltiazem  and verapamil , did not affect [Ca2+]i when applied to cones with ‘low baseline’ [Ca2+]i (<100 nM). This is exemplified in the double cone depicted in 2+]i and sensitivity to diltiazem  whereas the accessory member was unaffected by the drug . The average baseline in cones that did not respond to D-cis diltiazem was 42±5 nM (n = 24), not statistically significant from the cohort of total cones. In contrast, the average baseline [Ca2+]i in which D-cis stereoisomer decreased [Ca2+]i was 95±21 nM (n = 14), significantly different from healthy controls (P<0.001). Finally, exposure to diltiazem decreased baseline [Ca2+]i to 56±17 nM, not statistically different from healthy non-treated cones  antagonist thapsigargin . In 2 mM Ca2+-containing control saline, thapsigargin elevated [Ca2+]i levels to 85±13 nM. In such cones, a potentiated sensitivity of cytosolic [Ca2+]i for external Ca2+ entry was observed. Elevation of [Ca2+]o from 1 mM to 10 mM resulted in an approximately six-fold [Ca2+]i change in TG-treated cells, elevating cytosolic [Ca2+] from 42±6 nM to 245±33 nM  while lesser [Ca2+]i increases were observed in the ellipsoid region (241±35 nM (n = 12)  . The senectively .2+ entry that occurs in resting cones is masked by powerful extrusion systems mediated by the PMCA transporters 2+-permeable channels in hyperpolarized cones we employed the Mn2+ quenching protocol in cones loaded with the Fura-2 indicator dye. Mn2+ ions appear to traverse most Ca2+-permeable channels; upon entering the cell, Mn2+ irreversibly binds Fura-2 and quenches its fluorescence 2+ influx through L-type voltage-operated channels activated by depolarizing the cone with high KCl. Depolarization increased the Fura-2 emission ratio, exemplified by the opposite response to the 340/380 nm excitation wavelengths. Fluorescence elicited by both wavelengths was quenched by 100 µM Mn2+ whereas 10 µM ionomycin produced little additional quenching, indicating that most of the Fura-2 is confined to the cytosol of the cone inner segment. At the [Ca2+]-independent isosbestic excitation wavelength (360 nm), exposure to Mn2+ elicited quenching of Fura-2 fluorescence  from which Ca2+ spread into the cell body and the myoid/subellipsoid. Cytosolic [Ca2+] within the ellipsoid remained low, possibly due to mitochondrial uptake 2+ signals, the kinetics of the [Ca2+]i increase after induction of SOCE were slow (panel H). Second, a quasi-uniform [Ca2+]i increase was earliest observed across the nuclear area which is replete with ER cisternae 2+ elevation induced by SOCE diffused into the subellipsoid space (arrows in K) as a result of blocked SERCA-mediated Ca2+ sequestration, underscoring the role of the ER in regulation of subellipsoid and myoid [Ca2+]i. This data demonstrates that SOCE exhibits a distinct spatiotemporal signature that can be distinguished from previously reported modes of Ca2+ entry in cone photoreceptors.Cone SOCE is spatially represented in 2+ influx in ‘resting’ cones might be maximized by combination of hyperpolarization and increased [Ca2+]o. Cone inner segments in the salamander retinal slice were voltage-clamped and stimulated with voltage ramps ranging from −150 to 0 mV in the presence of 10 mM external Ca2+. After the Ih cation conductance was blocked with internal and external Cs+, little transmembrane current was observed in control saline. However, depletion of ER stores with thapsigargin combined with an increase in transmembrane Ca2+ gradient evoked significant currents  in the presence of Cs+. The hyperpolarization-evoked current was blocked by elimination of extracellular Ca2+  or Gd3+ (5 µM)   . The Ca2 µM Cd2+ . This da2+ overshoots in cones  . 2-APB (2-aminoethoxy-diphenylborate) (100–200 µM), another typical inhibitor of SOC channels, suppressed depletion-evoked Ca2+ signals by ∼70% . Application of another lanthanide, La3+ (10 µM), resulted in ∼80% inhibition  (Inhibition by low (µM) concentrations of lanthanides and organic antagonists such as SKF 96365 or 2-APB is considered to be diagnostic for SOC channels ; n = 3) . The mea= 0.027)  of the o2+ channels: the drug had no effect on the amplitude or the time course of voltage-operated Ca2+ currents , rods 2+, the universal voltage-activated Ca2+ channel antagonist, had no effect on the amplitude of Ca2+ overshoots. In 15 cells tested, peak SOCE was 237±83 nM, not significantly different from 299±122 nM in 100 µM Cd2+ , not significantly different from control values (data not shown). This suggests that depletion of ER stores does not modulate CNG-mediated Ca2+ signaling in cone ISs. Taken together, this data suggests that cone photoreceptors express a plasma membrane Ca2+-permeable pathway that exhibit a pharmacological profile characteristic of SOC channels.We next tested the effect of the CNG channel antagonist L-cis-diltiazem on SOCE. In the presence of 10 µM diltiazem, the amplitude of [Ca2+]i (>50 nM), sensitivity of [Ca2+]i baseline to D-cis-diltiazem and insensitivity to further depolarization  and verapamil (50 µM). Occasionally, depletion-evoked overshoots continued as sustained [Ca2+]i plateaus that were partially sensitive to L-type channel antagonists (2+ overshoot in 46% of cells (n = 17/37), suggesting that depolarization evoked during the induction of SOCE was insufficient for crossing the threshold for L-type channel activation. The drug suppressed the amplitude of the overshoot more strongly in cones in which [Ca2+]i baseline was already elevated; in contrast, Ca2+ overshoots in most (71%) of the cones that exhibited no change in baseline response to D-cis diltiazem, were also insensitive to the drug  evoked in horizontal or OFF bipolar cells by strong depolarizing steps  applied to simultaneously recorded presynaptic cones. This stimulus evokes a fast inward current due to activation of AMPA receptors followed by a very small, sustained inward current ed cones . This da2+ channels, equally suppressed the transient and sustained components of the postsynaptic EPSC  , an antagonist of L-type Ca) n = 4; . L-cis d) n = 4; , suggest2+ loading state of the ER store to plasma membrane store-operated TRPC and/or Orai channels Recent RNAi screening studies showed that SOCE is activated by the clustering of the E-F hand protein STIM1 in the ER underneath the plasma membrane 2+ influx mechanism that modulates the dynamic range of presynaptic Ca2+ signaling in vertebrate cones. While the mechanism operates maximally under light-adapted conditions it may also contribute to sustained signaling in the darkness. Unexpectedly, its activation is facilitated by depletion of ER Ca2+ stores, implicating SOCE in photopic signaling in the retina.We investigated a Ca2+ after depletion of Ca2+ stores in the cone inner segment transiently elevated [Ca2+]i to several hundred nM and was typically followed by a prolonged [Ca2+]i plateau. The pharmacological profile of this depletion-sensitive mechanism exhibited many hallmarks of SOCE, including sensitivity to the Ca2+ driving force and suppression by lanthanides and organic SOCE antagonists SKF-96365, 2-APB and MRS 1845. Irreversible inhibition of Ca2+ sequestration into the ER by thapsigargin both elevated baseline [Ca2+]i and potentiated the magnitude of Ca2+ transients generated by re-exposure to mM [Ca2+]o. Whereas micromolar concentrations of La3+ and Gd3+ decreased cytosolic baseline [Ca2+]i close to the levels observed in [Ca2+]o-free conditions, in hyperpolarized cells this effect was not duplicated by L-cis-diltiazem, D-cis-diltiazem, verapamil or Cd2+, suggesting that baseline [Ca2+]i transients are not maintained by either L-type nor CNG channels.Restoring extracellular Ca2+]i elicited by changes in the Ca2+ gradient was similar to concentration-dependence of Ca2+ “overshoots” observed in cells with depleted ER stores, suggesting that part of the standalone “resting” current in light-adapted cells derives from SOCE. Previous studies showed that a significant fraction of Ca2+ ions entering the cells through SOC channels is immediately pumped out of the cytosol by high-affinity PMCA transporters 2+ can enter the cells through store-operated channels despite the fact that these channels are less permeable to Mn2+ than to Ca2+2+ quenching of the Ca2+ indicator dye revealed substantial sustained divalent cation influx into the hyperpolarized cone IS. This cation influx was highly reproducible, observed in all studied cones, was insensitive to antagonists of voltage-operated channels yet antagonized by the SOCE blockers Gd3+ and MRS 1845.The half-maximal sensitivity for baseline Δ[Ca2+ entry through store-operated channels is likely to parallel the proposed inwardly rectifying cation conductance mediated by CNG channels 2+]i signals in the cone inner segment emphasized the somatic compartment. While differing markedly from [Ca2+]i gradients mediated by L-type channels in depolarized cones  is relatively low , limiting the dynamic range of presynaptic [Ca2+]i to ∼two-fold 2+ signal. Indeed, we found that steady-state [Ca2+]i in partially depolarized salamander cones is determined through intimate interactions between SOCE and voltage-operated Ca2+ entry. This conclusion was suggested by pharmacological manipulations of Ca2+ overshoots, plateau [Ca2+]i and Ca2+ oscillations by L-type and CNG channel antagonists in cells with depleted ER stores. However, in the majority of salamander cones, possibly corresponding to fully light-adapted cells, baseline [Ca2+]i tended to range between 30 and 70 nM. These levels were unaffected by antagonists of voltage-operated Ca2+ entry and were almost entirely sustained by Ca2+-permeable channels that were sensitive to antagonists of many store-operated and TRP channels. Hence, the dynamic range of average presynaptic [Ca2+]i in salamander cones appears to be at least ∼20–40 fold, ranging from ∼50 nM in the light to several µM measured in depolarized cone terminals 2+]i levels closer to the mouths of Ca2+ channels at the active zone are likely to be significantly higher in the tens of µM range We found that i as well as cone output signals. These signals may not only function in replenishment of ER stores, but also stimulate local Ca2+ pathways required for cone signaling and survival 2+ entry.Taken together, our data suggests that SOCE integrates signaling pathways in the plasma membrane  with functional state of the intracellular Ca2+]i concentration was measured in cone ISs loaded with the AM ester of the fluorescent indicator Fura 2  as reported previously Ambystoma tigrinum) were decapitated and pithed using procedures recommended by the National Institute of Health Guide for the Care and Use of Laboratory Animals. Retinas were dissociated in 0 Ca2+/papain  saline for 20 min at room temperature (20–22°C). Cells were plated onto coverslips coated with 0.2 mg/ml concanavalin A . The recording chamber was superfused via an electronically controlled multi-inlet manifold. The control saline solution contained, (in mM), 97 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 2 lactic acid, 0.3 ascorbic acid and 1 taurine at 240 mOsm. pH was adjusted to 7.6 with NaOH. To stimulate glycolysis, glucose concentration in the saline was elevated to 20 mM. All salts and STIM1 antibody were obtained from Sigma. 2-APB, L- and D-cis diltiazem were from Biomol ; verapamil, thapsigargin and SKF 96365 were from Tocris .i and provides large changes in signal for [Ca2+]i starting from resting levels in light-adapted photoreceptors 2+]i was calculated after subtraction of the background fluorescence by measuring the ratio of the two emission intensities for excitation at 340 and 380 nm. Numerical ratio information was exported into a data analysis program  and used to calculate the concentration of free [Ca2+]i in the cone cytosol.Isolated photoreceptors were loaded with 2–5 µM fura-2 AM for 10 minutes and washed for 20 minutes. This indicator has a dissociation constant (Kd) of 224 nM which is close to resting i. Fluorescence emission induced by both 340 nm and 380 nm excitation wavelengths was quenched by Mn2+ (1 (slope in Mn2+)/a2  ×100. Because these non-ratiometric measurements were affected by bleaching of the indicator dye (0) from first and last exposures to control saline, quantifying the effect of tested compounds as: [(a1−a0)/(a2−a0) ×100].For fura-2 quenching protocol, 50–100 µM Mn by Mn2+ . >95% of by Mn2+ . The ratator dye , the dat2+] levels were calibrated in vivo with 10 µM ionomycin in 0 and 10 mM [Ca2+]o saline using the standard relationship and a Kd for Ca2+ binding to fura-2 of 224 nM 2+]i cones are defined as having resting [Ca2+]i under 80 nM, typically around 50 nM. “High-baseline” cells are defined as having resting [Ca2+]i>100 nM. Data for cells which did not complete the calibration process is presented as 340/380 nm ratios. All pooled data is presented as mean±S.E.M. Significance was determined using the t-test .Free i elevation in the soma and the subellipsoid.(3.98 MB AVI)Click here for additional data file.Video S2Following store depletion, [Ca2+]i elevations are first observed in the cell body, followed by the synaptic and subellipsoid regions of the cone inner segment.(5.69 MB AVI)Click here for additional data file.