After deletion of the Ca2+ sensor for fast release – synaptotagmin-1 – slower Ca2+-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools  driven by alternative Ca2+ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca2+-dependent action: a Ca2+-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca2+-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca2+-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca2+-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca2+ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca2+-dependent properties of secretion without assuming parallel pools or sensors.Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca 2+-dependent fusion of vesicles with the plasma membrane. Kinetic heterogeneity is ubiquitous in secretory systems, with fast phases of release on the millisecond time scale being followed by slower phases. In the absence of synaptotagmin-1 – the Ca2+sensor for fast fusion – the fast phase of release is absent, while slower phases remain. To account for this, mathematical models incorporated several releasable vesicle pools with separate Ca2+ sensors. However, there is no clear evidence for parallel release pathways. We suggest a sequential model for Ca2+-dependent neurotransmitter release in adrenal chromaffin cells. We assume only a single releasable vesicle pool, and a Ca2+-dependent catalytic refilling process from a limited upstream vesicle pool. This model can produce kinetic heterogeneity and does better than the previous Parallel Pool Model in predicting the Ca2+-dependence of releasable pool refilling and the consequences of SNARE-protein mutation. It further accounts for the release in the absence of synaptotagmin-1 by assuming that the releasable vesicle pool is depleted, leading to slow and Ca2+-dependent fusion from the upstream pool, but through the same release pathway. Thus, we suggest that the elusive ‘alternative Ca2+ sensor’ is an upstream priming protein, rather than a parallel Ca2+ sensor.The release of neurotransmitter involves the rapid Ca The essential nature of Ca2+ for the final step of neurotransmitter release has been known since the pioneering work of Bernard Katz 2+-cooperativity of this step was demonstrated by Dodge and Rahamimoff 2+ uncaging made it possible to describe this cooperativity quantitatively and to derive mathematical models for the Ca2+ triggering step 2+-dependent 2+-dependent steps with respect to each other is currently less than clear.Neurotransmitter release and synaptic transmission depend on the fusion of secretory vesicles with the plasma membrane by exocytosis, and the ensuing release of the contained neurotransmitter molecules. Exocytosis itself is the conclusion of a number of steps, which starts by the generation of the vesicle and its filling with neurotransmitter, and continues with the transport of the vesicle to the plasma membrane, its physical attachment to the membrane (docking), the attainment of fusion competence (priming), and ends with its fusion as the result of the arrival of a Ca2+-dependent vesicle priming and neurotransmitter release through a sequential pathway with one release sensor 2+ channels. The notion of parallel releasable vesicle pools was reinforced when deletion of the Ca2+ sensor synaptotagmin (syt) (-1 or -2) was found to eliminate fast release, while Ca2+-dependent slow release components remained Initially, most working models assumed Ca2+ sensors for fast release in vivo and reconstituted in vitro, including SNAREs, Munc13/CAPS and Munc18 2+-dependent properties of regulated secretion.Despite the fact that parallel pathways have been the working model for more than a decade, molecular correlates of the slow release pathway are still missing: syt-1, syt-2 and syt-9 are now widely accepted to be Ca2+-dependent priming steps. Recent studies concluded that initial assembly of SNARE-complexes is catalyzed by Ca2+-sensitive molecules such as Munc13 2+-dependent catalytic mechanism in the strict sense, i.e. a mechanism, which increases both forward and backward rates by reducing the energy level of the transition state. In addition, to account for data obtained in syt-1 knockout cells (see below), we suggest that the Ca2+ sensor for fast release ensures the steady-state population of the Readily Releasable Pool (the RRP) at rest, either by preventing SNARE-dependent fusion in the absence and synchronizing fusion in the presence of Ca2+2+-unbound state Based on properties derived from molecular perturbation studies, we propose a sequential model, where we make novel assumptions regarding the nature of the Ca2+-dependence of secretion from chromaffin cells. Moreover, our model can account for recovery of the RRP after strong stimulation and deliver parsimonious explanations for the observed effects of SNARE or syt mutations without the need to evoke additional parallel pathways or release sensors. We suggest that a fresh look at sequential models is of value to understand kinetic diversity in secretory systems.By mathematical modeling and comparison to older and new data we here show that these assumptions suffice to explain the Ca2+-dependent properties of regulated exocytosis, we investigated a mathematical model with sequentially arranged pools , separated by Ca2+-dependent steps 2+-dependent, driven by a Michaelis-Menten Ca2+-dependent forward process (k1) with a Kd of 2.3 µM, as in previous models of the adrenal chromaffin cell 2+ speeds up their re-supply, as observed experimentally 2+-dependent catalysis. A catalyst lowers the activation energy barrier of a reaction (2) and backward (k−2) rates , where this had not already been done in the literature 2+ sensors for RRP and SRP fusion are separate sequential and cooperative sensors each binding three Ca2+ ions  using an abrupt Ca2+-step from 0.5 µM to 25 µM  remains high, giving rise to the sustained component of release. A particularity of the PPM is that fusion during the sustained phase is almost entirely through the SRP, since the SRP-to-RRP conversion is relatively slow and Ca2+-independent  pathway) Pioneering work to 25 µM . Both movesicles . Finally2+-dependent, driven by the Ca2+-dependent catalyst, giving rise to the slow burst phase. It should be noted that the identification of the fast component with the RRP and the slow component with the NRP is only approximate, because the size and kinetics of the two phases depend on the entire system , the fast burst component corresponds mainly to the rapid depletion of RRP-vesicles, whereas the slow burst component corresponds mainly to NRP-vesicles that mature to the RRP-state before fusing . This mae system . TherefoPool k1, . Other tSince both models could reproduce biphasic burst release and the sustained component, we next compared our new model with data that had previously been fitted with the PPM 2+ signal driving the release model allowed the determination of amplitudes and rate constants of fast and slow burst components as a function of pre- and post-flash Ca2+ concentrations  through release before the uncaging event. However, the relative proportion of fast and slow components remained fairly constant . We also constructed a version of the model with a cooperativity of two , as well – as previously shown 2+-independent. This Ca2+-independence was concluded from the observation of parallel enhancement of the fast and slow burst by increasing steady state [Ca2+]i in the sub-micromolar range before stimulation 2+-dependent acceleration of priming is catalytic. Therefore, the two models can be distinguished by probing the recovery of the RRP under conditions of elevated Ca2+: according to the SPM, its recovery should be sped up, whereas the PPM predicts Ca2+-independent - and thus slower - recovery.One fundamental difference between the PPM and the SPM is the recovery behavior of the RRP after its depletion. In the PPM, the refilling of the RRP from the SRP is Ca2+-uncaging had emptied both the fast and the slow burst components 2+ uncaging flash at variable inter-stimulus intervals (ISI) while measuring Ca2+-relaxation  and a change in k3 is expected to affect both fast and slow release. This is demonstrated in 3 (lines are model simulations). Moreover, the relative contribution of fast and slow release depends on the time constant of fast release  unaffected. In contrast, in the SPM model, the amplitudes and release rates are all functions of the elementary rate constants kd k3 see . Therefo release , reducin3 . Mutagenesis studies of layer +8 in synaptobrevin-2 previously performed by us 3  in particular.It is commonly assumed that one of the last events before vesicle fusion is the assembly of the final interaction ‘layer’ (layer +8) in the SNARE complex. In the SPM this molecular event is primarily reflected by the rate constant k2+-dependent catalyst with a downstream Ca2+ sensor predicts the effects of SNARE mutations.Thus, a sequential model featuring an upstream Ca2+ sensor in various systems slow release components persist. This is also the case in mouse adrenal chromaffin cells, where deletion of syt-1 leads to ablation of the fast burst component, whereas the slow burst component and the sustained component both remain 2+ sensor (even though no such molecule has been found yet) is an easy and reasonable way to account for this finding. How can one account for it in a sequential model?One argument for parallel pool or parallel sensor models has been the observation that after deletion of the fast Cain vitro and in vivo experiments have emphasized the function of syts and/or complexins as clamps on release 2+-binding to syt would release the clamp and allow the SNARE complex to complete its assembly, leading to rapid membrane fusion. In our model, the clamp would generate the final energy barrier for fusion (2+ ions to syt-1 (4)  indirectly led to refilling of the RRP through a Ca2+-independent process  assumed that Ca process . A Ca2+- at rest . If mere2+-dependent catalyst. This in effect makes both the forward and the reverse reactions Ca2+-dependent. Moreover, because the catalyst acts on the transition state . Moreover, the Ca2+-dependent NRP-to-RRP conversion speeds up RRP refilling, which makes it possible to explain the faster recovery kinetics upon strong stimulation  where the RRP size appears to be limited by a fixed number of ‘release sites’.It is not necessary for the validity of our model that a protein can be found that only acts catalytically without inducing pool size effects. Instead, the catalyst may be a molecule on the plasma membrane, the association to which is necessary for priming, but which is sped up by Ca2+ binding kinetics than syt-1 2+ sensor itself, because asynchronous release was still Ca2+-dependent when Doc2α was mutated to be Ca2+-insensitive 2+-dependent proteins driving priming. The alternative hypothesis of another syt-like molecule, taking the place of syt-1 or syt-2 in their absence, is also possible, but additional Ca2+-dependent priming reactions need to be assumed to account for the enhanced recovery of the RRP during elevated Ca2+ , which makes it easy to derive pool behavior. Future efforts will be needed to understand whether a sequential model when combined with spatial heterogeneity has explanatory power in the world of neurons.When considering neurons, other factors will contribute to kinetic diversity of release. Chief amongst these is the variable distance between RRP vesicles and Ca2+ sensitivity of vesicle supply from the Depot. We also used the parameters of the release sensor found for the RRP vesicles (three site binding model 2 and RRPCa3 (and Depot) sizes at rest, where RRPCan represents RRP vesicles with n Ca2+ bound.To model the release from chromaffin cells we set up the reaction rate equations, which consist of a set of ordinary differential equations describing the temporal change in the population of each state (see below). We followed the suggestion by Thomas Voets 2+-dependent catalyst. Therefore, the rate constants 2k and −2k are functions of the Ca2+ concentration. A catalyst acts by increasing the rate of a reaction through the lowering of the activation energy barrier (20k and −20k). We suggest that the overall rate constants 2k and −2k are functions of the un-catalyzed (Ca2+-independent) rate constants 20k and −20k and functions of the faster, catalyzed (Ca2+-dependent) reaction rates 2catk and −2catk. The relative contribution of the catalyzed rate in turn depends on the probability (g(Ca2+)) of the catalyst to have bound the required number of Ca2+ ions in order to become activated. For simplicity, we assumed that the supply of catalyst is not limited . The overall rate constants 2k and −2k take the following form:2+ to the catalyst, so that the catalyst is in equilibrium with Ca2+ at all times. In general, a catalyst might bind n Ca2+ ions in order to be activated:2+-binding step:n of Ca2+ ions) to total catalyst (g) is2+-levels are well defined. g(Ca2+) can be rearranged:n Ca2+ can bind independently and with identical dissociation constants:g(Ca+2) can be further simplified:2+-cooperativity of one (n = 1). Then 2+), the following relationship holds true:20k to −20k ratio), the uncatalyzed re-supply rate of vesicles (20k) and the asymptotic rate of NRP-to-RRP conversion at high Ca2+-levels to calculate all four rates.We suggest that the transition between the NRP and RRP states is catalyzed by a Ca barrier . Yet a cD and the 2catk values of the catalyst from the previous study by Thomas Voets  = 0.5 and the slow component has reached 50% of its asymptotic rate . As can be seen from the data points in 20k was calculated by using 2+ concentrations (500 nM), the rate 2k is equal to 0.12 s−1−20k and −2catk were calculated using −2k at resting Ca2+ concentration is 0.1 s−1 and at steady state the 2k/−2k ratio is 1.2/1 We estimated the K1Maxk = 55 fF/s is a constant because of the assumption of constant Depot size The kinetic equations for the model presented in 0A was fixed to the baseline value immediately before the onset of the Ca2+ step and 0t was set to the inflection point of the cumulative release after stimulus onset. All other parameters were free. 1A and 1τ correspond to the amplitude and time constant of the fast, and 2A and 2τ to the size and time constant of the slow burst component, respectively. 3A is the slope of the sustained component and t is time. The rate constants displayed in The sizes and time constants of the fast and slow release components were determined by fitting a sum of exponentials to 5 s of simulated cumulative release. The sustained release that is typically observed after the burst phase of secretion was approximated by a line. The fit function used had the following form:2+ signal at the site of the vesicle in response to the depolarization is not known. In order to test whether our model in principle would allow for a selective depletion of the fast- without major depletion of the slow component, we resorted to a simplified stimulus and probed the effect of a Ca2+ step from 0.5 µM to 25 µM on the NR and R states. In order to mimic the experimental conditions of the previous study we were looking for a point in time after onset of the stimulus, where the majority of fast vesicles were depleted, whereas the majority of the slow component's amplitude was still left intact, which was satisfied at the condition shown in the top panel of 2+ was then stepped back to resting (0.5 µM) levels and the system was allowed to re-equilibrate for various inter-stimulus intervals before a second step to 25 µM Ca2+ was applied. Then, the sizes and time constants of the two components were again obtained by the fitting of exponentials (see above).The temporal change of the two kinetic components during recovery of the fast component of release was characterized and compared to the experimental values presented in 2+ levels were similar in all conditions, allowing us to solve the model's differential equations under the assumption of constant 2k, −2k and 3k (F) contribute to the cell's increase in membrane capacitance (MC) during secretion.For the comparison between the PPM and the SPM, the data presented in 2 and k3 . TherefototV. We further assumed that the NRP and RRP states are in equilibrium and that the fusion rate is essentially zero (3k = 0) before stimulation, such that:MΔC, which is equal to F(t) when calculating in capacitance units) in terms of the kinetic rate constants 2k, −2k and 3k (same rate constants as depicted in st second) of release from chromaffin cells expressing wildtype and mutant synaptobrevin-2, using a custom made macro in Igor Pro 6.22A (Wave Metrics). The onset of the capacitance increase was determined manually.To arrive at the initial conditions we assumed a fixed amount of vesicles in the burst phase (the sum of [NRP] and [RRP]), 2k and −2k while varying 3k. The values of 2k and −2k were determined by a global fit of the model to the data sets shown in 2+ uncaging (from 0.5 µM to 25 µM) for 5 s while varying the fusion rate (RRPk = RRP1/τ) in the following model:RRP]0 = 61.3 fF and [SRP]0 = 52.3 fF.The data of the measured time constants and release amplitudes following mutation of synaptobrevin-2 shown in Figure S1Fit of experimental data from chromaffin cells with cooperativity 2 for the calcium catalyst. Same data as in 2+- catalyst for vesicle priming has cooperativity 2 for Ca2+. A. The rate constants for fast and slow burst release. The model with cooperativity 2 displays more saturation in the slow rate constant at high [Ca2+]i B. The sum of the fast and slow component , or the fraction of fast- to total burst release  are fairly insensitive to post-flash Ca2+C. The amplitudes of the fast- and slow release components are a bell-shaped function of the resting Ca2+-levels Click here for additional data file.Figure S2Under strong stimulation, the recovery of the fast component is sped up in the SPM, but the slow component recovers even faster. Same experiment and simulation as in (EPS)Click here for additional data file.Figure S32+-dependent catalyst from the SPM to speed SRP-to-RRP interconversion (PPM+cat).Evaluation of an extended model based on the PPM, but with the incorporated Ca Same data as in A. The top panel shows the full model based on the PPM with the additional Ca2+-dependent rate constants, modeled as a catalyst on the SRP-to-RRP reaction , or the fraction of fast- to total burst release  are fairly insensitive to post-flash Ca2+C. The amplitudes of the fast- (blue) and slow (red) burst components are a bell-shaped function of the resting Ca2+-levels Click here for additional data file.Table S1Model parameters for the Sequential Pool Model (SPM) with cooperativity 2 for the catalyst See  for fits(DOC)Click here for additional data file.Table S2Model parameters for Parallel Pool Model incorporating a catalyst Click here for additional data file.


Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca2+ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.Inositol 1,4,5-trisphosphate receptors (IP They allow release of Ca2+ from intracellular stores in response to the many stimuli that activate phospholipase C 3R subtypes (IP3R1-3), and most cells from vertebrates express functional IP3R that are homo- or hetero-tetrameric assemblies of these IP3R subtypes and their splice variants 3R diversity is poorly understood, and nor are there ligands that usefully discriminate between IP3R subtypes. It is, however, clear that activation of IP3R is initiated by binding of IP3 to the conserved IP3-binding core  of each IP3R subunit 3R in native cells make it difficult to define unambiguously the functional properties of each IP3R subtype. Stable heterologous expression of mammalian IP3R in the only vertebrate cell line engineered to lack all endogenous IP3R (DT40 KO cells) 3R subtype to define structure-activity relationships for key endogenous and synthetic inositol phosphates 3R subtype with adenophostin A  and its most important analogues Inositol 1,4,5-trisphosphate receptors  may be a more important determinant of the increased affinity of AdA for IP3R AdA, originally isolated from 3R activation and associated changes in cytosolic Ca2+ signalling 3R subtypes. The need for such analyses is particularly important in attempting to explain results in which Ca2+ signals evoked by IP3 differ from those evoked by AdA 3R have been genetically inactivated 3R subtypes and thereby define structure-activity relationships for AdA and its key analogues for each IP3R subtype.The high-affinity and metabolic stability of AdA have generated considerable interest in both the synthesis of AdA analogues and their application to analyses of IP3 was from Alexis Biochemicals . AdA manno-AdA and xylo-AdA Sources of most reagents were provided in a previous publication 3R subtype or react equally with all three subtypes, we established that in the DT40 cells used, levels of IP3R expression (relative to IP3R3) were IP3R1 , IP3R2 (48±5%) and IP3R3 (100%) 3R expression for each cell line, and differences  may affect both the size of the IP3-sensitive Ca2+ pool and its sensitivity to IP33R expression do not compromise the analyses reported here, which are entirely concerned with relative potencies of AdA analogues for each IP3R subtype (see below).From quantitative analyses of western blots using antisera that selectively recognise each IP2+] within the endoplasmic reticulum of permeabilized DT40 cells was provided in preceding publications 3R subtypes was loaded with a low-affinity Ca2+ indicator (Mag fluo-4) 2+ accumulation, which was monitored at intervals of ∼1 s using a FlexStation 3 fluorescence plate-reader . CLM had the following composition: 140 mM KCl, 20 mM NaCl, 1 mM EGTA, 20 mM Pipes, pH 7, free [Ca2+] ∼220 nM (after addition of MgATP), and carbonyl cyanide 4-trifluoromethoxy-phenyl hydrazone  to inhibit mitochondrial Ca2+ uptake. After 150 s, when the stores had loaded to steady-state with Ca2+, IP3, AdA or its analogues was added with thapsigargin (1 µM) to prevent further Ca2+ uptake, and after a further 30 s, the response was recorded. Agonist-evoked Ca2+ release was expressed as a fraction of that released by ionomycin (1 µM) A comprehensive description of the methods used to measure free [Ca2+ stores released by maximally effective concentrations of agonist, and pEC50 values (-log EC50) were calculated. For convenience some results are presented as EC50 values, but all statistical comparisons use pEC50 values. Within each experiment, the pEC50 for AdA was determined to allow paired comparisons with values obtained for each AdA analogue. These are reported as ΔpEC50, where:Concentration-effect relationships were fitted to Hill equations using GraphPad Prism (version 5.0) from which Hill coefficients (h), the fraction of the intracellular Ca50 values, like those shown in We note that post hoc test, as appropriate, with P<0.05 considered significant. Because not all comparisons of the relative potencies of AdA and IP3 were paired, the SEM of this ΔpEC50 value was calculated from:p is the estimate of the population variance:Statistical comparisons used Student’s t-test or ANOVA followed by Bonferroni’s 1 and s2 are the sample standard deviations, and n1 and n2 are the sample sizes 3-evoked Ca2+ release from intracellular stores are unexpectedly complex. It is widely observed that under conditions where Ca2+ uptake into the endoplasmic reticulum (ER) is inhibited, submaximally effective concentrations of IP3 rapidly release only a fraction of the IP3-sensitive Ca2+ stores 3 on the rate of Ca2+ release. The mechanisms underlying this pattern of response, known as quantal Ca2+ release 3R as the Ca2+ content of the ER declines 3-senstive Ca2+ stores 2+ release evoked by submaximal concentrations of either IP3 or AdA from permeabilized DT40-IP3R1 cells is quantal. These observations provide the justification for all subsequent experiments in which the concentration-dependent effects of IP3 or AdA were measured 30 s after their addition , ribophostin (in which a methoxy group replaces the adenine moiety of AdA) and furanophostin (in which only the furanoid ring remains) . Maximalthan AdA  and 2. T3 requires the adenine moiety of AdA positioned to allow it to form a cation-π interaction with Arg-504 in the α-domain of the IBC of IP3R1, a residue that is conserved in all IP3R subtypes 3R is likely to be similar for all IP3R subtypes.These results are consistent with our earlier conclusion that the 10-fold greater affinity of AdA relative to IP2OH and 2″-OH substituents of the glucose ring of AdA are thought to mimic the 3-OH and 6-OH of IP3, respectively  in the IBC 33 (2′-phosphate of AdA) are in different rings in AdA  3 and the 5″-CH2OH of AdA IP2) caused its potency and affinity for IP3R1 to decrease by ∼100-fold 3R1  of the clam-like IBC, closure of which initiates IP3R activation 3All known active analogues of IP2+ release via any IP3R subtype  is close to the threshold for detecting Ca2+ release (4″-dephospho-AdA at concentrations up to 300 µM failed to evoke Ca subtype . This is potency . The syn release .3-sensitive Ca2+ store is likely to be due solely to its reduced affinity rather than reduced efficacy. A concentration of 3″-dephospho-AdA (30 µM) that caused detectable Ca2+ release via IP3R1 (∼21±5%) had no effect on the sensitivity of the Ca2+ release evoked by a subsequent addition of IP3. The pEC50 was 7.00±0.02 and 7.04±0.06 (n = 3) for IP3 alone and in the presence of 3″-dephospho-AdA, respectively  close to that of IP3  and the 5-phosphate contacts the other side (α-domain) 3R share this essential 4,5-bisphosphate moiety. AdA is different in that its 4″-phosphate  and the α-domain 3 are important for IP3 binding, the equivalent structures within AdA play lesser roles.A key step in the initial activation of IPaffinity . We sugg2+ entry, which is triggered by depletion of IP3-sensitive Ca2+ stores 2+ signals have been reported to be differentially affected by IP3, AdA or its analogues 3R subtypes, suggest that different physiological effects of IP3, AdA or its analogues are more likely to result from differences in their affinities, kinetics or rates of degradation than from selective interactions with different IP3R subtypes.Both store-operated Ca


While higher Ca2+ binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca2+ affinity by 27% in the steady-state measurement and increased the Ca2+ dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca2+ sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca2+ sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca2+ sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca2+ sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent.The Ca These changes produce strong, force-generating interactions between actin and myosin. The Ca2+-induced disruption of the cTnI-actin interaction is modulated by the PKA phosphorylation of cTnI, feedback from strong actin-myosin interaction and is also is highly dependent on the sarcomere length The cardiac troponin (cTn) trimeric complex plays an essential role in regulating cardiac contractility. It consists of a highly conserved CaMutations within the cTn complex can alter both contraction and relaxation of the heart and may lead to cardiomyopathies 2+ binding remain unclear. We previously demonstrated that the L29Q substitution occurs naturally in cold water fish, and postulated that along with three other teleost-specific substitutions , they play a role in the higher Ca2+ sensitivity involved in adaptation to lower core temperatures Several studies have investigated the molecular mechanisms of this mutation 2+ binding properties and myofilament Ca2+ sensitivity of cTn in multiple systems with increasing complexity. These include cTn complexes, reconstituted thin filaments and ultimately, single skinned cardiomyocytes with the native cTn complexes replaced by recombinant cTn complexes. Different cTn complexes that were used include: mouse wild type cTnC or mutant L29Q cTnC, wild type cTnT, and WT cTnI or SD cTnI. Molecular dynamics simulations were also performed to model the structural changes in the WT & L29Q cTnC/cTnI complex in response to the phosphorylation of the cTnI1–32 cardiac specific extension.This study investigates whether the L29Q cTnC mutation abrogates the effects of PKA-dependent phosphorylation of cTnI on cTnC Ca2+ affinity and in combination with pseudo-phosphorylation of cTnI, extends this effect. When measured in the context of skinned cardiomyocytes, the L29Q mutation yields a small increase in Ca2+sensitivity of force, but appears to reduce the effect of phosphomimetic mutations. This effect is most pronounced at shorter sarcomere length (SL), yet it is abrogated with the presence of pseudo-phosphorylation of the cTnI N-terminal extension.The experiments presented herein revealed that the effect of the L29Q mutation varies with the context of the experiments. This variability suggests a greater degree of complexity in cTn function than has been encapsulated in previous work. We show here that the neither the L29Q mutation nor pseudo-phosphorylation has any significant impact on the cTnI/cTnC complex when it is isolated from the thin filament. However, when the L29Q cTnC is incorporated into thin filament preparations the L29Q mutation decreased cTnC CaSee E. coli. BL21(DE3) (Novagen) cells. The cTnC gene was in the ampicillin resistant pET21a vector, cTnI and cTnT were in in the kanamycin resistant pSBET and pET24a vectors respectively. Expression and purification of cTnC, cTnI, and cTnT were carried out using methods described in Dong et al. and Kruger et al.Recombinant mouse cardiac protein subunits were expressed in The method for cTnI was modified as follows: The TnI-containing cell lysate was made to 25% saturation with ammonium sulphate, and subject to centrifugation at 27,000 g for 30 mins (Beckman JLA16.25) at 4°C. The supernatant was dialysed overnight against 2–3 changes of 4 liters of buffer A  and loaded onto a CM sepharose column  previously equilibrated with buffer B . Cardiac TnI eluted at 40–50% of a linear gradient of 0–0.5 M NaCl in buffer B. If required, as determined by SDS-PAGE, cTnI was dialyzed against buffer B and further purified using a DEAE fast flow column . Cardiac TnI was then dialyzed extensively against 5 mM ammonium bicarbonate, lyophilized, and stored at −20°C.T53C and reconstitution of the Tn complexes were prepared as described by Davis et al.T53C and L29Q cTnCT53C was measured during Ca2+ titration with an excitation wavelength of 330 nm and an emission wavelength of 450 nm. For these experiments the free Ca2+ concentration was calculated using EGCA02, a program developed by Robertson and Potter Fluorescent labeling of cTn2 were added to 2 mL of each labeled cTn complex (0.15 µM) and reconstituted thin filaments (0.15 µM) in a titration buffer  with constant stirring. Each titration measurement was at least duplicated and four to five datasets were collected for each cTn complex for each thin filament preparation. The data were fit with a Hill-equation 2+ affinity for each cTn complex was represented by the dissociation constant Kd ± SEM at the half-maximal fluorescence change p<0.05.Microliter amounts of CaCl2+ dissociation rate constants (koff) were measured at 15°C using a stopped-flow apparatus with a dead time of 1.4 ms . The rates of conformational changes induced by EGTA removal of Ca2+ from labeled cTn complexes or thin filaments were measured. The samples were excited at 330 nm and the emission was monitored through a 420–470 nm band-pass interference filter for the cTn complexes and a 510 broad interference filter for the thin filaments. Each koff value represents an average of at least three separate experiments with each averaging at least five traces fit with a single exponential equation. The buffer for the stopped-flow measurements was in mM: 10 MOPS, 150 KCl, 3 MgCl2, 1 DTT, pH 7.0 at 15°C.Reconstituted thin filaments were prepared using protocols described previously 2 5.70, Na2ATP 6.31, EGTA 2, potassium propionate 156, BES 10, DTT 1, leupeptin 0.1, PMSF 0.1, pH 7.0) containing 0.3% Triton-X for 4 minutes. The myocyte pellet was washed twice using cold relaxing solution. Finally, the SCM were re-suspended in relaxing solution and kept on ice during the day of the experiment.Ventricular myocytes were obtained by enzymatic digestion of hearts from C57BL/6 female mice (3 to 4 mos.) et al.2, and 1.0 DTT at 4°C. The SCM were bathed in the extraction buffer containing either cTnT-WT cTnI or cTnT-SD cTnI for approx. 20 min at 15°C. The SCM were then washed twice with the relaxing solution. The maximal Ca2+-activated force was measured before and after cTnT-cTnI treatment to determine the extent of endogenous cTn removed. A maximal force decrease ≥90% was used as a criterion for cTn extraction, indicating high endogenous cTn 2+-activated force was measured to determine the extent of cTn reconstitution and SCM with >80% force recovery was used as an inclusionary criterion and was not significantly different between the recombinant cTn complexes.Cardiac troponin complex substitution was performed using a modification of Chandra The apparatus used to measure SL and force from SCM and for rapid solution changes has been described previously 2+-activated force (F) with preset SL as the reconstituted myocyte was exposed to a series of solutions with varied Ca2+ concentrations (pCa 4.5–8.0) 2+ sensitivity was determined in separate experiments by carrying out two force-pCa measurements for each SCM with the SL of the myocytes set to 1.9 µm and subsequently to 2.3 µm.The steady-state force-pCa relationship was determined by measuring the CaKF1/2 which represents the Ca2+ sensitivity and corresponds to the [Ca2+] required for generating half-maximal force. The data were analyzed statistically using Student’s t-test and one-way repeated measures analysis of variance (One way ANOVA). The values of KF½ were reported as means ± SEM. Two means were considered to be significantly different when the p<0.05.Each force-pCa curve was fit to the Hill equation using Origin 8.5 as described before 1–32 peptide (PDB: 2JPW) on the WT cNTnC+switch peptide cTnI 147–163 (PDB: 1MXL) was performed on the ClusPro 2.0 server Docking of the cTnIFmax <1000.0 kJ mol−1 nm−1 and equilibrated for 1 ns with a time step of 0.002 ps, with position restraints placed on all atoms of the protein and peptide. Interactions were calculated using the Particle Mesh Ewald method T value of 0.1 and a τP value of 1.0. The simulation box was periodic in all dimensions. After equilibration, the position restraints on the protein atoms were replaced with P-LINCS bond length constraints and bond angle restraints Fmax of 250.0 kJ mol− 1 nm− 1. Analyses of the simulations were carried out using Visual Molecular Dynamics Each protein system was embedded in a cubic box with 9 Å between the edge of the protein and the edge of the box and solvated using the SPC-E water model. The net charge of the system was made zero by replacing randomly selected solvent molecules with Na or Cl ions. The solvated system was energy minimized using the steepest descents algorithm to an The formation and the stability of both the cTn complex and the reconstituted TFP were individually verified using stopped-flow kinetics. The complexes were found to be stable in the absence of free cTnC, and free cTn complexes were not detected in the reconstituted TFP. The abbreviations for the various constructs used in the different preparations are listed in 2+-dependent IAANS fluorescence decrease was observed in all four cTn complexes and no significant differences in the steady-state Ca2+ binding or dissociation parameters were observed (2+ binding affinity of different cTn complexes became evident upon titrating the reconstituted TFP with Ca2+. A Ca2+-dependent fluorescence increase was observed for each of the four TFP configurations. The Kd of TFP containing the SD cTnI (SD cTnT53C) was 1.2 fold higher than TFP containing the WT cTnI (WT cTnT53C) (p<0.05), indicating that the pseudo-phosphorylated cTnI significantly decreased the Ca2+ affinity of cTnC in the TFP. Unexpectedly, the Kd of TFP containing L29Q cTnC (L29Q cTnT53C) was ∼1.2 fold higher than that of the WT (p<0.05), suggesting that TFP containing L29Q cTnC had a lower affinity for Ca2+ (A). This is in contrast to the observed increase in Ca2+ binding affinity in isolated L29Q cTnC T53C) as a function of Ca2+ concentration, TFP that include L29Q cTnC produce a curve with a statistically significant rightward shift of 0.08 pCa units relative to that of SD cTnT53C with WT cTnC (p<0.05) (B), indicating that L29Q further reduced the Ca2+ affinity.A measurable Caobserved . Changesfor Ca2+ A. This (p<0.05) B, indicnH) of all the cTn complexes were within the range of 0.91–0.99 and no significant differences were observed among the different cTn complexes. The values of the Hill coefficients of the TFP were, as anticipated, much higher overall. The Hill coefficient of the TFP containing WT cTn was significantly higher when the pseudo-phosphorylated SD cTnI was included in the complex. The presence of the L29Q cTnC mutation significantly (p<0.05) decreased the Hill coefficient by 34% and 23% for the WT cTnI and SD cTnI containing complexes, respectively. These data reveal that the L29Q mutation has an effect on Ca2+ regulation in TFP containing either the WT or the phosphomimetic cTnI.2+ koff value 1.6 fold higher than that observed for WT (p<0.05). In TFP containing WT cTnI, the inclusion of L29Q cTnC did not appear to have any effect; however in SD TnI containing TFP, the presence of the L29Q mutation further increased the Ca2+ dissociation rate concomitant with the effect of the pseudo-phosphorylated cTnI.Examination of the effect of phosphomimetic TnI mutations in the context of TFP yielded a Ca2+ activated force changes before and after extraction with the cTnI·cTnT complex. Chandra et al. observed a Ca2+ activated force lost at 85–90% in detergent skinned left ventricular papillary muscle fiber bundles after cTnI·cTnT treatment. By running analytical PAGE on these skinned fiber bundles, they estimated that at least 80% of the endogenous cTnI was extracted Due to the difficulty in running analytical PAGE on single skinned cardiac myocytes, the efficacy of native cTn complex exchange with the recombinant cTn complex was determined by measuring the maximal Ca2+ sensitivity have been studied extensively 2+] (pCa) in SCM reconstituted with either WT cTn or L29Q cTn. The force-pCa curves of SCM reconstituted with L29Q cTn were left shifted, indicating an increase in myofilament Ca2+ sensitivity. The Ca2+ sensitivity for L29Q cTn increased 1.2-fold (p<0.05) in SCM (2+ sensitivity 1.4-fold compared to WT cTnC The effects of PKA-dependent phosphorylation of the N-terminus of cardiac troponin I (cTnI) on myofilament Ca) in SCM . This fiT53C IAANS reporter was used and SCM experiments in which no reporter was used, control experiments were performed in SCM. In these experiments, cTnCT53C IAANS reporter was used in additional reconstitution experiments of SCM with the WT cTnC and L29Q cTnC constructs  Reconstitution of SCM with troponin complexes incorporating the cTnCT53C IAANS reporter had a slight but significant effect of Ca2+ desensitization. As can be seen in T53C IAANS and L29QcTnT53C IAANS had significantly higher KF1/2 values than their respective non-reporter controls. 3) In the comparison of the L29Q cTnC with and without the IAANS reporter in SMC, the L29Q mutation consistently had a Ca2+ sensitizing effect and the L29QcTnT53C IAANS exhibited significantly (p<0.05) lower mean KF1/2 values than WT cTn T53CIAANS (Because of the difference in the results seen in TFP in which the cTnCnstructs . Three o53CIAANS .B). The Ca2+ sensitivity of force generation of SCM reconstituted with SD cTn was 1.38-fold lower than that of SCM with WT cTn. These results are consistent with previous findings which showed that phosphorylation of Ser23/24 reduced the Ca2+ affinity of cTnC thereby increasing the rate of muscle relaxation 2+ sensitivity of force generation for SCM containing either WT cTnI or SD cTnI was increased when L29Q cTnC is present  in SCM, the force-pCa curves were shifted to the right of those of SCM reconstituted with recombinant WT cTn which included WT cTnC and WT cTnI B. The C2+ sensitivity with sarcomeric stretch. These results corroborate the well-documented length-dependent myofilament Ca2+ sensitivity F1/2 values for the SD cTn at both SL 2.3 µm and 1.9 µm were significantly higher than that for the WT cTn at both SL (p<0.05), indicating that SD cTnI rendered the SCM less sensitive to Ca2+. This phenomenon is more pronounced at shorter SL (1.9 vs. 2.3 µm), demonstrating a significant increase in the length-dependent myofilament Ca2+ sensitivity. In addition, the SCM reconstituted with L29Q SD cTn had a higher KF1/2 at SL 1.9 µm that was significantly different from both WT and SD (p<0.05); however, at SL 2.3 µm, the KF1/2 of L29Q SD cTn was significantly different from WT (p<0.05) but was not different compared to SD cTn. This resulted in a much shallower slope of the KF1/2 value and SL relationship in SCM containing L29Q SD cTn than those containing SD cTn and WT cTn , docked with the cardiac specific N-terminal extension of cTnI1–32 (PDB: 2JPW) with and without pseudo-phosphorylation .After 100 ns of simulation, the major structural changes within the triplex were the position of the cTnI switch peptide and the N-terminal extension of cTnI2+ binding affinity nor the Ca2+ dissociation rates were different between the WT cTnT53C and L29Q cTnT53C complexes. Similarly, no significant difference in Ca2+ binding affinities or kinetics of isolated Tn complexes containing pseudophosphorylated cTnI and either TnCT53C or L29Q cTnCT53C were observed. While these results were not entirely expected, they suggest that a more complex system that includes both actin and tropomyosin is required to study the interactions between troponin subunits 2+ dissociation from cTn though it caused a small but significant decrease in Ca2+ binding affinity. However, in TFP reconstituted with SD cTnI, the presence of the L29Q mutation further enhanced the increase of the Ca2+ dissociation rate caused by pseudo-phosphorylation of cTnI. These results convincingly demonstrated that at the thin filament level, the L29Q cTnC mutation alters the ability of Ca2+ binding to cTnC and affects the functional effects of the phosphomimetic cardiac troponin I. It has been well-documented that phosphorylated cTnI decreases the Ca2+ binding affinity of cTnC by increasing the koff to facilitate muscle relaxation In the comparison of the effect of the L29Q cTnC mutation in isolated Tn complexes, neither the Ca2+ binding affinity resulting from the L29Q cTnC mutation observed in the thin filament preparation was not commensurate with a reduction in Ca2+ sensitivity of force generation in SCM. Instead, the L29Q cTnC mutation resulted in a significant increase in Ca2+ sensitivity of myofilament activation (similar to what we observed previously with cTnC reconstitution 2+ desensitization effect of SD cTnI. One of the possible reasons for these divergent results may be related to the effects of the cTnC mutations (C35S/T53C/C84S) designed for the incorporation of the cTnCT53C IAANS or the IAANS itself we used as a Ca2+ reporter in the in vitro experiments on TFP in comparison to the experiments without the IAANS reporter in the reconstituted SCM. Previous studies have shown that these cTnC mutations and subsequent labeling with IAANS behave biochemically and physiologically similar to wild-type and endogenous cTnC 53C/IAANS demonstrate that while the engineered reporter is not completely innocuous on SCM function, the sensitizing effect of the L29Q cTnC has approximately equal magnitude of −0.46 and −0.45 µM in the presence and absence of the IAANS reporter’ respectively. Thus the data support the notion that the differences in observations on the effect of L29Q cTnC between the thin filament preparation and the skinned cardiomyocyte are distinct from the uniform sensitizing effect of the cTnCT53C/IAANS reporter that is observed in the WT and L29Q experiments.To understand better the effect of the L29Q cTnC mutation in a system closer to physiological conditions, the native cTn complexes were exchanged with various recombinant cTn complexes containing both the L29Q and WT cTnC in SCM. The decrease in CaTwo other major differences between TFP and SCM that could contribute to the differences in results we obtained are the impact of: sarcomere length and the presence of myosin and other proteins of the thick filament. While these are not completely mutually exclusive concepts, SL will be discussed first.2+ sensitivity of force induced by the L29Q cTnC mutation affected length dependent activation (LDA). The present study in which SCM were reconstituted with troponin complexes corroborates that finding. Thus it is important to note that the effect of the L29Q cTnC mutation on Ca2+ sensitivity of force generation was more pronounced at shorter SL (1.9 µm) than at longer SL (2.3 µm) (A–D), which resulted in a significant decrease in the slope of the relationship between Ca2+ sensitivity of force and the SL (E). Interestingly, a similar reduction in SL-dependence of contractile activation caused by enhanced Ca2+ binding of cardiac troponin was also recently reported by Korte et al.2+ affinity and reduced the SL-dependence of myofilament Ca2+ sensitivity. This finding has several important implications: 1) the L29Q cTnC mutation should impact LDA that is an important component of the Starling relationship of the heart; 2) the high myofilament Ca2+ sensitivity of the L29Q cTnC mutation at the shortest SL (1.9 µm) which corresponds to near peak systolic conditions could alter Ca2+ dynamics within the cardiomyocyte with the potential of being arrhythmogenic 2+ sensitivity with the L29Q cTnC mutation and others which did not in vitro studies provide no means of controlling or assessing the length or the conformation of the thin filament which could clearly mask such findings. The multicellular skinned fiber preparations likely pose a problem due to heterogeneity of SL within the preparation and lower values of reconstitution with the recombinant troponins compared to single cardiomyocytes used in the present study. The latter implies higher retention of endogenous cTnC or troponin complex. In the present study, the L29Q cTnC mutation induced a significant increase in Ca2+ sensitivity in SCM reconstituted with the recombinant mouse cTn at sarcomere lengths of 1.9 and 2.1 µm but not 2.3 µm. However, by replacing endogenous cTnC with recombinant human L29Q cTnC or WT cTnC in skinned fiber bundles from left ventricular papillary muscle, Dweck et al.et al.et al.2+ sensitivity. Gollapudi et al. reported a small but significant decrease in myofilament Ca2+ sensitivity by exchanging the endogenous cTn with homologous recombinant cTn et al.2+ sensitivities in skinned fibers reconstituted with L29Q cTnC were statistically insignificant with respect to the WT, they did observe tendencies to increase the Ca2+ sensitivity of the mutant that led them to conclude that the disparity was attributable to the increased sensitivity of the single SCM system, compared to their skinned multi-cellular cardiac muscle fiber technique. Indeed, single SCM have distinct advantages over skinned cardiac muscle fiber bundles. The shorter diffusion distances in skinned cardiomyocytes will make troponin subunit exchange more effective and avoid the concentration gradient build-up of substrates (ATP) and end products (Pi and ADP) during contraction et al. (∼70%) et al. (∼60%) 2+ sensitivity caused by the cTnC mutants. More importantly, cardiomyocytes are short and thin enough for one to precisely determine SL changes, which is a crucial parameter to be controlled in determining the effects on myofilament Ca2+ sensitivity. Thus, the discrepancies between our study and the aforementioned studies may be due to the differences in SL when determining Ca2+ sensitivity in skinned cardiac preparations. The study of Gollapudi et al.et al.et al.Our previous study using the cTnC exchange technique in the skinned cardiomyocytes (2.3 µm) , A–D, whd the SL E. Inter2+ sensitivity exists 2+, one would expect an increased probability of strong cross bridge formation and hence increased myofilament Ca2+ sensitivity. Experiments on myofilaments using high molecular weight compounds (e.g. dextran or mannitol) as a means of increasing osmotic compression and causing a reduction in myocyte diameter have demonstrated increased Ca2+ sensitivity et al.et al.2+ cooperativity including: the influence of length on the interaction between adjacent troponin-tropomyosin complexes on the thin filament 2+ sensitivity of force generation. The presence of the other myofilament proteins, such as myosin, that causes the feedback effects of cross-bridge is likely another reason for the different effects of the L29Q cTnC mutation in our reconstituted TFP and SCM.While length dependent activation is an important physiological principle, particularly in heart muscle, considerable controversy as to the mechanism by which LDA increases myofilament Ca2+ activation was altered by the effect of cross-bridges. The presence of other myofilament proteins that cause the feedback effect of bound cross-bridges on L29Q cTnC-cTnI-Tm complex might also play an important role in determining the Ca2+ sensitivity of force generation in SCM.The above hypothesis is supported, at least in part, by the changes in the Hill coefficients. Studies have shown that the activation of myocardial contraction is highly cooperative, and the alterations in cooperativity are typically, but not always, manifested as a change in the Hill coefficients 2+ in cardiac muscle. Although the reduction in SL-dependence of contractile activation caused by the L48Q cTnC mutation in the recent study by Korte et al.2+ activation in cardiac muscle and corroborates our hypothesis.The L29Q cTnC mutation seems to perturb the interaction between the cTnI N-terminus and the cTnI switch region and eventually exerts its effect on the interaction between cTnI and actin. This effect may disturb the feedback effects of cross-bridges on the interaction between cTnC-cTnI-Tm complex and actin. Studies have shown that cross-bridge binding to the thin filaments can increase the apparent affinity of cTnC for Ca2+ activation has been shown to be affected by PKA-mediated phosphorylation of cTnI. In the present study, SD cTnI caused an expected reduction in Ca2+ sensitivity of force generation with a more pronounced and significant decrease in Ca2+ sensitivity at SL 1.9 µm than at 2.3 µm. Konhilas et al.2+ desensitization was also greater at shorter (1.9 µm) SL, resulting in a steeper relationship between Ca2+ sensitivity of force and the SL 2+ sensitivity of force more profoundly at shorter SL, which reduced the increase in the slope of Ca2+ sensitivity-SL relationship caused by SD cTnI. These results further support our hypothesis that the alterations in cTnC-cTnI interaction cause more pronounced changes in Ca2+ sensitivity of force at shorter SL, thus affecting the length dependence of myofilament Ca2+ activation.Several studies have postulated that the major impact of the L29Q cTnC mutation is on its effect on the interaction of PKA-phosphorylated cardiac troponin I (cTnI) with cTnC and its ability to impact thin filament regulation et al. found that S23/24D cTnI reduced pCa50 at both SL, primarily at 2.3 µm, resulting in a reduced SL-dependence of Ca2+ sensitivity. The source of this discrepancy is not clear; however, it is likely due to the experiment conditions, heterogeneous proteins, and the difference between the skinned cardiac muscle preparations used for the studies as discussed below. Previous studies have shown similar inconsistent results when an increase in SL dependence of Ca2+ sensitivity was found in PKA treated skinned cardiac myocyte preparations whereas a decrease was reported in skinned cardiac trabeculae phosphorylated by PKA treatment.In contrast to our results, Rao et al.2+-desensitization, when assayed by incubating the cardiac muscle fiber bundles with phosphatase or PKA to phosphorylate sarcomeric PKA target proteins , and titin). PKA phosphorylation of the N-terminus of cTnI and MyBP-C may suppress cardiac contractility 2+ sensitivity in skinned rat ventricular trabeculae. Therefore, the phosphatase and PKA treatments of sarcomeric PKA substrates in skinned cardiomyocytes might affect Ca2+ sensitivity of force generation masking the effects of the L29Q mutation on Ca2+ activation.Neulen 2+ release from cNTnC thereby aiding the muscle relaxation process (11). Conversely, in the presence of the L29Q cTnC mutation, the pseudo-phosphorylation of cTnI does not cause any significant change in the opening/closing of cNTnC. The L29Q cTnC mutation renders the cNTnC in a more “open” conformation compared to the constructs containing WT cTnC especially when cTnI is pseudo-phosphorylated  and is virtually abolished at longer SL (2.3 µm). In other words, the L29Q cTnC mutation significantly blunted the length-tension relationship in this preparation which likely has consequences on the Frank Starling relation in vivo. Very recently this was also observed in a study using a wide variety of hypertrophic cardiomyopathy mutations in humans 2+ sensitivity in the presence of pseudo-phosphorylation of cTnI. The effects of the L29Q mutation on Ca2+ sensitivity of force may be caused by its interference on the interactions between thin filament proteins which alter the feedback effects of cross-bridges on the cTnC-cTn/Tm complexes. The small increase in myofilament Ca2+ sensitivity might not alter the contractility of cardiac muscle significantly during the daily activities of the patients with this cTnC mutation. However, during β-adrenergic stimulation where increased cardiac output is required, the mutation might affect pump function by reducing the length dependence of Ca2+ activation and the response of cardiac troponin to increased cardiac output. These effects are likely hypertophic in response to defects in contractility when physiological demand is increased. Our results are consistent with the postulate 2+ sensitizing effect of FHC-related cTnC mutants becomes more pronounced in the presence of PKA-mediated phosphorylation of cTnI induced by β-adrenergic stimulation but this effect is clearly sarcomere length dependent.The L29Q cTnC mutation decreased the Ca


Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca2+. The sources of the Ca2+ increase are from the endoplasmic reticulum (ER) Ca2+ pools as well as from Ca2+ influx. The store-mobilization component of the Ca2+ increase induced by the pHi rise was not sensitive to antagonists for either IP3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), leading to depletion of the ER Ca2+ store. We further showed that the physiological consequence of depletion of the ER Ca2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca2+ leak from ER pools followed by Ca2+ influx via SOCs.Intracellular pH (pHi) and Ca However, these intrinsic buffering systems can be overpowered during continued extra- and intracellular stress or stimulation. Cells, therefore, have evolved a complicated proton transporting system to regulate cytosolic pH as well as the pH in other cellular compartments The activity of virtually all proteins and macromolecules can be modulated by protons; thus intracellular pH (pHi) is rigorously regulated for survival +-H+-exchangers (NHEs) and the Na+-HCO3− co-transporters (NBCs), to extrude the protons produced during normal cellular metabolic activity. Some cells also utilize Na+-dependent Cl−-HCO3− exchangers (NDCBEs) or monocarboxylate-H+ co-transporters (MCTs) for similar purposes. On the other hand, activation of the Cl−-HCO3− anion exchanger (AEs) and plasma membrane Ca2+-ATPase (PMCAs) can lead to cytosolic acidification. Among all of these transporters, NHE1 is perhaps the most dominant one for maintaining pHi homeostasis +, but also by various extra- or intra-cellular signals, leading to elevation of pHi during diverse cellular processes, such as autophagy, migration, adhesion, chemotaxis, and cell cycle progression Under physiological conditions, cells utilize two major pH-regulatory ion transporters at the plasma membrane, the Na2+ is equally important in regulating diverse cell functions, including fertilization, cell proliferation and differentiation 2+ level at rest is kept low mainly by the active extrusion of cytosolic Ca2+ out of cells via the PMCAs and the Na+/Ca2+ -exchanger, as well as by sequestration of Ca2+ into the endoplasmic reticulum (ER) and mitochondria via a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and a mitochondria Ca2+ uniporter, respectively. External signaling can markedly increase cytoplasmic Ca2+ levels by opening plasma membrane ion channels, such as voltage-gated Ca2+-selective channels (CaVs) and transient receptor potential (TRP) ion channels. Cytoplasmic Ca2+ concentration can also be suddenly and dramatically increased by release from the ER Ca2+ store, through activation of IP3 receptors (IP3Rs) and ryanodine receptors (RyRs) in ER 2+ mobilizing messengers have been identified for regulating these Ca2+ release channels, which include inositol trisphosphate (IP3), nicotinic acid adenine dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Ca2+ influx and internal Ca2+ store release are normally interconnected. Thus, depletion of the ER Ca2+ store can trigger activation of the store-operated channels (SOCs) at the plasma membrane, mediated by Orails and Stims, and lead to Ca2+ influx Likewise, Ca2+ events. Intracellular Ca2+ spikes that occur during oocyte maturation and egg fertilization in several marine invertebrate and amphibian species are believed to be responsible for regulating the subsequent alkalinization of pHi 2+ uptake 2+ current in a wide variety of cells and tissues, including various types of neurons2+ channels are involved in alkaline pHi-triggered Ca2+ entry depending on cell or tissue types 2+ release are sparse and conflicting, especially concerning whether or how IP3 signaling is required Intracellular alkalinization has been linked to intracellular Ca2+ changes in various cell lines. We demonstrated that intracellular alkalinization inhibits SERCA activity, leading to Ca2+ leak from the ER. The depletion of ER Ca2+ stores then activates Ca2+ influx through SOCs of Stim1 and Orai1.Here we studied the mechanisms underlying intracellular alkalinization-induced cytosolic Ca4Cl) or methylamine, can induce cytosolic Ca2+ increase , an organic base commonly used in the organic chemistry, can induce cytosolic Ca2+ increases, similar to that of NH4Cl, in a dose dependent manner, whereas sodium acetate, a weak acid, failed to change Ca2+ , an IP3R antagonist, or U73122, a specific inhibitor of phospholipase C, had little effect on cytosolic Ca2+ increases induced by DIEA.HBr  to selectively disrupt the lysosomal membrane 2+2+ pools, evidenced by the fact that subsequent addition of GPN (50 µM) or bafilomycin A1 (0.5 µM), a specific inhibitor of the vacuolar-type H(+)-ATPase that is known to be able to release Ca2+ from the lysosomes normally, failed to release any more Ca2+. In contrast, pretreating cells with GPN failed to significantly alter the DIEA.HBr-induced Ca2+ rise in HeLa cells (Fiure 2D), indicating that the Ca2+ pools targeted by DIEA.HBr are not the lysosomes.To test whether the acidic Ca2+ release from ER by intracellular alkalinization markedly differed from that induced by histamine, which is known to be mediated by IP3Rs, whereas it is similar to the thapsigargin- triggered Ca2+ release  was markedly inhibited as compared to that in neutral (pH 7.2) or acidic (pH 6.4) media  media . Additiochondria . All the2+ to ER following Ca2+ release triggered by histamine or ATP in HeLa cells. In the absence of extracellular Ca2+, histamine releases Ca2+ from the ER into the cytosol via the IP3Rs, and SERCA then pumps the cytosolic Ca2+ back to the ER and returns the cytosolic Ca2+ to the basal levels. Thapsigargin abolishes SERCA activity, thereby greatly decreasing the decay rate of the cytosolic Ca2+. As showed in 2+ release was 11.1±0.9 nM/second (n = 36 cells), while this rate decreased to 4.9±0.3 nM/second (n = 29) in the presence of 10 µM thapsigargin. Similarly, the decay rate of histamine in the presence of DIEA.HBr was 5.8±0.4 nM/second (n = 9), which was also significant lower than that of histamine alone. Moreover, the effects of intracellular alkalinization on the decay rate of cytosolic Ca2+ after histamine treatment were examined in the presence of sodium orthovandate, a PMCA inhibitor  were significantly reduced compared to that in the acidified or neutral pH buffers . Thus, extracellular alkalinization also triggers cytosolic Ca2+ release from the ER Ca2+ pool, as well as induces Ca2+ influx. In addition, addition of 2 mM Ca2+ in Ca2+ free alkaline extracellular buffer induced Ca2+ increase through influx, which was markedly inhibited in NIH3T3 cells with Stim1 or Orai1 knockdown  on a conserved aspartate residue locks SERCAs in an E1 state bound with Ca2+, while the dephosphorylation of the residue, catalyzed by a conserved TGES motif of the actuator-domain switches SERCAs to a E2 state bound with protons, making them ready for exchanging with cytoplasmic Ca2+ and starting the next cycle. In cytoplasm, the protonation of a glutamate of the TGES motif is essential for the dephosphorylation of SERCA. It is thus reasonable that intracellular alkalinization could inhibit SERCA by preventing the protonation of the glutamate of the TGES motif at its cytoplasmic region. Inside the ER lumen, four carboxylate residues involved in Ca2+ binding in the E1 state are also needed to be protonated in order to release the bound Ca2+ and transit to the E2 state 2+ binding-carboxylate groups. This would result in inhibiting counter-transport of protons from inside ER lumen for the cytosolic Ca2+. Either way could lock SERCA in the phosphorylated E2 state and stop the pump cycle. Indeed, alkaline pH has been shown to reduce the rate of SERCA dephosphorylation in vitro 2+ at alkaline pH was decreased in vitro The molecular mechanisms of intracellular alkalinization induced inhibition of SERCA can be understood from its crystal structures 2+ entry via SOCs mediated by Orai1 and Stim1 in a wide variety of cells, yet whether alkaline pH directly regulates Orai1 and Stim1 remains to be determined. We have actually found that Ca2+ influx triggered by thapsigargin is not affected by alkaline pH  were maintained in DMEM (Invitrogen) plus 10% fetal bovine serum (Invitrogen) and 100 units/ml of penicillin/streptomycin (Invitrogen) at 5% CO2 and 37°C. PC12 cells (ATCC) were maintained in DMEM plus 7.5% horse serum, 7.5% fetal bovine serum, and 100 units/ml of penicillin/streptomycin at 7.5% CO2 and 37°C. The medium was changed every 48 h.HeLa, NIH3T3 and 293T cells (4 cells/well in regular medium overnight. Cells were then incubated with 1 µM BCECF AM (Invitrogen), an intracellular pH indicator, in Hank's balanced salt solution (HBSS) at room temperature for 30 min. Afterwards, cells were washed once with HBSS, and pHi of the cells in HBSS at room temperature was measured in TECAN Infinite® 200 plate reader in triplicates with excitations set at 440 nm and 490 nm and emission collected at 530 nm every 3 or 10 second. Emission ratios of two different excitations (490 nm/440 nm) were calculated. In addition, standard intracellular pH curve was obtained by Nigericin/High K+ method. Briefly, cells in different wells were incubated in calibration buffer  with different pHs, 6.6, 7.0, 7.6 and 8.1. The linear pH standard curve was created with defined buffer pH as X axis and fluorescence ratio (490 nm/440 nm) as Y axis. The intracellular pHs of cells treated with or without drugs were then obtained by calibrating the corresponding 490/440 ratio against the standard curve.HeLa cells were cultured in 96-well plates at the density of 2×104 cells/well in regular medium overnight and were labeled with 4 µM Fura-2 AM (Invitrogen) in HBSS at room temperature for 30 min. The cells were then washed with HBSS three times and incubated at room temperature for another 10 min. Cells were put on the stage of an Olympus inverted epifluorescence microscope and visualized using a 20× objective. Fluorescence images were obtained by alternate excitation at 340 nm and 380 nm with emission set at 510 nm. Images were collected by a CCD camera every 3 or 6 seconds and analyzed by a Cell R imaging software.Cells were cultured in 24-well plates at the density of 7×102+ uptake experiments were performed as described previously 2) containing 2 mM EGTA. HeLa cells were then permeabilized with 50 µg/mL saponin in intracellular buffer with 2 mM EGTA. Saponin at this concentration only selectively permeabilized the cell membrane while keeping ER membrane intact. The permeabilization efficiency was checked with trypan blue staining, which was typically over 95%. Next, the cells were washed twice in intracellular buffer to remove saponin and EGTA. Finally, permeabilized HeLa cells were suspended in uptake buffers. Uptake buffers were intracellular buffer plus ATP regeneration system  with pH adjusted with HCl/KOH to 6.4, 7.2, or 8.0. Fluo-3 salt (4 µM) was added to uptake buffers to monitor the calcium change. Fluo-3 fluorescence of permeabilized cells in different uptake buffers at 37°C was measured in TECAN Infinite® 200 plate reader with excitation at 488 nm and emission at 526 nm. The fluorescence bleaching was corrected by subtracting the control curve containing no cells.Castim1 and orai1 gene: CCCTTCCTTTCTTTGCAATAT and CACAACCTCAAC TCGGTCAAA, respectively. Then the two 21-mers were separately subcloned into pLKO.1, a replication- incompetent lentiviral vector for expressing shRNA. Scramble shRNA construct was used as a negative control. 293T cells were used to produce shRNA lentivirus as described previously 5 cells/well in 6-well plates. Next day, 120 µl of lentiviruses of stim1 shRNA, orai1 shRNA, or scramble shRNA were added to the cells in fresh medium containing 8 µg/ml polybrene. After 24 hrs, cells were selected in fresh medium containing 3 µg/ml puromycin for one week. Knockdown efficiency was verified by quantitative real-time RT-PCR or Western blot analysis.Two optimal 21-mers were selected in the mouse 5′ TCCCTGGTCAGCCATAAGAC and the reverse primer is 5′ TCATGGAGAAGGGCATAAGG. Forward primer for GAPDH is 5′ GGACGCATTGGTCG CTGG and reverse primer is 5′ TTTGCACTGGTACGTGTTGAT.The quantitative real-time RT-PCR using the iScript™ One-Step Kit With SYBR® Green (Invitrogen) was performed normally in Bio-Rad MiniOpticon™ Real-Time PCR Detection System according to the manufacture's instructions. The forward primer for orai1 is 5 cells/well in 6-well plates. Next day, cells were lysed in ice-cold EBC lysis buffer . Then the lysates were passed through a 21-gauge needle several times to disperse any large aggregates. Protein concentrations of the cell lysates were determined by the Bradford assay. Proteins (40 µg per lane) were diluted in the standard SDS-sample buffer and subjected to electrophoresis on 10% SDS polyacrylamide gels. Proteins were transferred to an Immobilon-P blotting membrane (Millipore), blocked with 5% milk in Tris-buffered saline  with 0.1% Tween 20, and incubated with primary antibody against Stim1  for 2.5 h. After washing, the blots were probed with a secondary antibody for detection by chemiluminescence.Control or shRNA-infected NIH3T3 cells were plated at density of 3×10pStim1-mCherry and pOrai1-EGFP, were provided by Dr. Gwack, Y 5 cells/well. Next day, pStim1-mCherry and pOrai1-EGFP were co-transfected into HeLa cells by Lipofectamine™ 2000. 48 hours after transfection, cells were washed twice with Ca2+ free HBSS. Distributions of Stim1 and Orai1 in transfected cells at room temperature were then examined in Ca2+ free HBSS containing thapsigargin or DIEA.HBr by confocal laser-scanning microscopy (Olympus FV300) with an Olympus PlanApo 60× Oil objective.Two plasmids, Figure S1Purification of DIEA.HBr. (A) HPLC fractionation of an esterification reaction. DIEA.HBr was purified in fraction 7. (B) Chemical structure of DIEA.HBr. (C) DIEA.HBr crystals were obtained by recrystalization method. (D) H-NMR of DIEA.HBR. (E) C-NMR of DIEA.HBr.(PDF)Click here for additional data file.Figure S22+ change compared with the effect of DIEA.HBr (4 mM).NaBr (4 mM) and KBr (4 mM) cannot induce any cytosolic Ca The graphs represent data from three independent experiments.(PDF)Click here for additional data file.Figure S3DIEA.HBr induces cytosolic pH increase in a dose dependent manner in HeLa cells. The graphs represent data from three independent experiments.(PDF)Click here for additional data file.Figure S42+ from ER pools in various cell types, including D3 mouse embryonic stem cells (A), NIH3T3 fibroblasts (B), BHK21 fibroblasts (C), HEK293T cells (D), HL 60 leukemic cells (E), PC 12 cells (F), Jurkat T lymphocyte cells (G), and THP-1 leukemic cells (H).Intracellular alkalization induced by DIEA.HBr (4 mM) releases Ca The graphs represent data from three independent experiments.(PDF)Click here for additional data file.Figure S5The effectiveness of Xestospongin C, U73122, and ryanodine. (A) Histamine (10 µM) induced Ca2+ rise was markedly inhibited by Xestospongin C  and U73122 . (B) Ryanodine  blocked caffeine (10 mM) induced Ca2+ rise in PC 12 cells whereas caffeine failed to induce Ca2+ increases in HeLa cells.(PDF)Click here for additional data file.Figure S62+ pools by GPN (50 µM) in HeLa cells.Complete depletion of lysosomal Ca Fura-2 loaded HeLa cells were treated with GPN (50 µM) to released lysosomal Ca2+. Subsequent addition of GPN (50 µM) or bafilomycin A1(0.5 µM) failed to release any more Ca2+.(PDF)Click here for additional data file.Figure S7Inhibition of SERCA ATPase activities by alkaline buffers in vitro. Top: HEK 293T cell lysates (300 µg) were incubated with anti-SERCA3 antibody  pre-bound to protein G beads. The SERCA3 immunocomplexes were then washed by TBS and separated evenly into three different pH Tris buffer (100 mM) at pH 7.5, 8.5, and 9.5, respectively. The ATPase activity of the immunocomplexes in different pH buffers were finally measured by a colorimetric assay for ATPase (Innova Bioscience) in a 96-well format and done in triplicates. As a control, boiling the immunocomplexes completely killed the ATPase activity. The graphs represent data from three independent experiments, and data quantification are presented as mean ± S.D., n = 3. Bottom: Western blot analysis of SERCA3 in SERCA3 IP complexes in indicated buffers after ATPase assay.(PDF)Click here for additional data file.Figure S82+ pool in HeLa cells.Intracellular alkalinization induced by DIEA.HBr decreases ionomycin-releasable Ca After 7 min of DIEA.HBr (4 mM) or MQ pretreatment, ionomycin (5 µM) was used to examine intracellular Ca2+ pool content in Ca2+ free HBSS containing 2 mM EGTA. The graphs represent data from three independent experiments. Quantifications of ionomycin-induced Ca2+ peaks were expressed as mean ± S.E., n = 30–50 cells, p<0.05.(PDF)Click here for additional data file.Figure S92+ uptake capability in HeLa cells in uptake buffer containing ruthenium red.Alkaline pH inhibits thapsigargin-sensitive Ca Quantifications of Fluo-3 fluorescence at 25 min after drug additions were expressed as mean ± S.D., p<0.05. All graphs represent data from three independent experiments.(PDF)Click here for additional data file.Figure S102+ store filling after histamine treatment in the presence of a PMCA inhibitor.Intracellular alkalinization inhibits ER Ca (A) Thapsigargin (10 µM) or DIEA.HBr (4 mM) inhibited ER Ca2+ refilling after histamine (10 µM) treatment in HeLa cells in the presence of sodium orthovandate (5 mM), a PMCA inhibitor. (B) HeLa cells were first over-loaded with Ca2+ by extracellular Ca2+ (2 mM) and ionomycin (10 µM) addition. Then the buffer was changed to Ca2+ free HBSS, and the removal of over-loaded intracellular Ca2+ was significantly inhibited in the presence of sodium orthovanadate (5 mM) compared with that in control.(PDF)Click here for additional data file.Figure S112+ pools in HeLa cells.Intracellular alkalinization partially depletes ER Ca Treating the cell with DIEA.HBr (4 mM) around the peak of Ca2+ curve induced by thapsigargin (10 µM) failed to cause additional Ca2+ rise, whereas adding thapsigargin (10 µM) around the peak induced by DIEA.HBr (4 mM) triggered another Ca2+ rise. The graphs represent data from three independent experiments.(PDF)Click here for additional data file.Figure S122+ content and subsequent cytosolic Ca2+ increase in HeLa cells.The partial inhibition of SERCA by lower doses of thapsigargin results in partial depletion of ER Ca (A) ER Ca2+ concentration, indicated by the thapsigargin (10 µM)-induced Ca2+ increase, was inhibited by pretreatment of Fura-2 loaded HeLa cells with 10 nM thapsigargin. (B)10 nM thapsigargin also directly induced cytosolic Ca2+ increase in HeLa cells.(PDF)Click here for additional data file.Figure S13Intracellular alkalinization has no affect on SOCE pathway induced by thapsigargin. In Ca2+ free HBSS, thapsigargin (10 µM) was used to completely deplete ER Ca2+ pool, then intracellular alkalinization was induced by applying DIEA.HBr (4 mM). The amplitude of Ca2+ influx during intracellular alkalinization exhibited no significant differences compared with that in control. The graphs represent data from three independent experiments.(PDF)Click here for additional data file.


It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca2+ release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca2+ release from intracellular stores in HEp-2 cells caused by ascorbic acid and antimycin A. In the presence of rotenone, antimycin A stimulates ROS production by mitochondria. Ascorbate-induced Ca2+ release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca2+ release in HEp-2 cells caused by ascorbic acid is associated with induced mitochondrial ROS production. The data obtained are in line with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets.Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca Redox processes involving transfer of electrons or hydrogen atoms are central processes of energy conversion in respiratory organisms. Recently, it has become apparent that numerous functionally significant biological processes proceed with participation of physical mechanisms ensuring intermolecular electron transfer. Electron transfer between low-molecular weight components of cytosol and intracellular proteins leads to the change of a functional state of both cellular proteins and cells as a whole , 2. All  in vitro, ascorbate cytotoxicity (EC50 < 4 mM) was observed in many types of cancer cell lines, whereas normal cells were resistant cyt) were performed as previously described cyt in human larynx carcinoma HEp-2 cells accompanied by depletion of intracellular thapsigargin-sensitive calcium stores [2+ release, from mitochondria and inositol-1,4,5-triphosphate-responsive Ca2+ stores. To examine involvement of mitochondria in ascorbate-induced Ca2+ release we used rotenone, mitochondrial complex I inhibitor. It was shown that treatment of cells with rotenone led to the decrease of ascorbate-induced Ca2+ release in HEp-2 cells  is the primary free radical in mitochondria, which is formed as the result of electron “leak” from the electron transport chain elements to oxygen [1 or cytochrome c should decrease electron flow from ubiquinol to Rieske iron-sulfur cluster resulting in a rise of ubisemiquinone concentration and superoxide production. Dismutation of O2∙− by the mitochondrial matrix Mn-superoxide dismutase leads to the formation of H2O2 [2O2, in turn, can oxidize thiol groups of targets and regulate Ca2+ release [2O2 by ascorbate in the extracellular solution catalase was used. Ascorbate-induced Ca2+ release from mitochondria was shown to be unaffected by catalase in an concentration of 500 U/mL (not shown). Thus, the important point in our model is that ROS production by ascorbic acid proceeds nearby specific redox sensors.It is generally accepted that superoxide anion  used in the study  from NADH to ubiquinone, the former being faster than the latter. Recent observations have led to the conclusion that rotenone enhances ROS formation during the FET and inhibits it during the RET [ψ. Generation of O2∙− and H2O2 by the electron transport chain is amplified by an increase in Δψ [ψ [ψ [2+ release  and ubiquinol: cytochrome c oxidoreductase (complex III) . In our  the RET . Moreovese in Δψ . The inhin Δψ [ψ . PreviouΔψ [ψ [ψ . In such release  and anti release . The resin cells ; therefonhibitor . Taken tShc has been shown to localize in the mitochondrial intermembrane space and redox cycle with cytochrome c to produce H2O2 that induces permeability transition [Shc pathways may in part explain enhancement of ROS generation. Further investigation will improve our understanding of mechanisms by which mitochondria are stimulated to produce oxidants and what the proximate targets of such oxidants are. The data of this preliminary study point out that complex III may be a possible player of ROS production in mitochondria under ascorbate treatment. It is important to emphasize that multiple sources of ROS generation have been identified in mitochondria , 28. Recansition . In cond2+ signaling —appears. The important implication of mitochondrial enzymes in redox regulation is that in this case signal decoding in cells depends also on the “transducer” activity in different types of cells. Thus the redox modulation of Ca2+ sparks occurs in species- and tissue-specific fashion. Some agents (antimycin A) may increase the ROS production and Ca2+ response in cells, while others (rotenone) decrease it (see 2+ sparks that are sensed and decoded into defined cell actions by a broad variety of cellular effectors.On the other hand, the results obtained indicate a new possible way of redox regulation of Caignaling . Calciume it see . Local R2+ release proceeds as a result of local effects of mitochondrial oxidants. In general, results of our research are in agreement with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets . Our obsEarlier was supposed that selectivity of ascorbate effect on cancer cells was mediated by an altered acid-base balance in tumor tissues . Changes


Isoforms 1–4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca2+ around 30 nM  or around 400 nM . Zebrafish GCAP isoforms showed also differences in their Ca2+/Mg2+-dependent conformational changes and in the Ca2+-dependent monomer-dimer equilibrium. Direct Ca2+-binding revealed that all zGCAPs bound at least three Ca2+. The corresponding apparent affinity constants reflect binding of Ca2+ with high (≤100 nM), medium (0.1–5 µM) and/or low (≥5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca2+-sensor system in zebrafish rod and cone cells supporting a Ca2+-relay model of differential zGCAP operation in these cells.Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca Light causes a drop of intracellular Ca2+, which triggers activation of membrane bound guanylate cyclases (GC) in rod and cone cells. This effect is mediated by GCAPs and has mainly been studied with the mammalian isoforms GCAP1 and GCAP2 Neuronal calcium sensor proteins regulate different neuronal functions by interactions with specific targets Fugu rubripes) Cyprinus carpio) Danio rerio) A larger variety (six to eight) of GCAP isoforms was found in pufferfish , CATATGGGTCAGCGGCTCAGCG-3′5′-TTC and GAATTCAAAAGTTGGCGCTGCGGC-3′5′-GTG (zGCAP2), CATATGGGCGCCCACGCAT-3′5′-GGA and GAATTCACTCCTTC-3′5′-GTTCCCGTCCAC (zGCAP3), CATATGGGGGACTCCTCCAGCAT-3′5′-CTC and GAATTCATGCTTGATCCTCGATGATC-3′5′-AGT (zGCAP5), CATATGGGCCAGAATCAAAGCGATG-3′5′-AAA and GAATTCATGTTTTCTTCCCCAAGTTGT-3′5′-AAA (zGCAP7). For heterologous expression in E.coli the amplified PCR product was ligated into a pET-expression vector using NdeI and EcoRI restriction sites. Plasmid preparation, restriction analysis and transformation were done according to standard protocols. Sequences of all zGCAP forms were verified by DNA sequencing.The cloning of zGCAP4 has been described elsewhere E.coli cells. Expression was started at an OD600 of 0.6 by induction with IPTG and bacteria were grown for 4 hours at 37°C. Bacteria were harvested by centrifugation and stored until use at −80°C. After thawing and cell lysis zGCAP forms were isolated from the soluble cell fraction (zGCAP3 and 4) or from inclusion bodies . Purification was a combination of anion exchange chromatography on a HiLoad 26/10 Q sepharose-column and of size exclusion chromatography on a HiLoad 26/60 Superdex 75-column (both from Amersham). The columns were equilibrated in 20 mM Tris-HCl pH 7.5, 1 mM DTT and NaCl in different concentrations. Before the samples were applied to size exclusion chromatography proteins were precipitated by ammonium sulfate to reduce volume. Prior to chromatography steps insoluble proteins from inclusion bodies were completely denatured using 6 M guanidine hydrochloride and refolded during dialysis against chromatography buffer  including one buffer change.Large amounts of non-myristoylated zGCAPs were obtained by overexpression in BL21 50-values the zGCAP concentration was varied between 0.25 and 10 µM. Ca2+-dependent GC activities were obtained by keeping zGCAP concentrations fixed at 10 µM and varying the free [Ca2+] using a K2H2EGTA/CaH2EGTA buffer system as described previously 2+-concentration was 1 mM. All incubation steps were performed under very dim red light, the reactions were stopped and further processed for analysis as described.Guanylate cyclase activity in bovine rod outer segment (ROS) membranes originates mainly (ca. 96%) from ROS-GC1 and to a lesser extent (4%) from ROS-GC2 2 or EGTA to a final concentration of 1 mM. The reaction was stopped after 2, 5 or 10 minutes by boiling and further analyzed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis.GCAP samples were treated with trypsin similarly to Purified protein samples (20 µg) were analyzed for the presence of monomeric and/or dimeric zGCAP forms by employing size exclusion chromatography on a BioSep-SEC S2000 column  including molecular mass standards as described 2, 1 mM DTT) at a final concentration of 0.7–1.2 µM. The excitation wavelength was set to 280 nm, the emission spectrum was recorded from 300–420 nm. Different free [Ca2+] were adjusted by a set of Ca2+/EGTA buffer solution (see above).Intrinsic Trp fluorescence measurements were performed with a spectrometer from photon Technology International (pTI) essentially as described before 2+-binding to zGCAPs was monitored using a previously described chelator assay 2+ between the protein and a chromophoric chelator whose absorbance changes upon Ca2+ binding. Similar amounts (10–50 µM) of the chelator 5,5′Br2-BAPTA and protein are titrated with Ca2+ and the binding of the cation to the chelator is monitored by UV absorbance at 263 nm. Data were fitted by employing a Newton-Raphson direct least-square fitting procedure implemented in the CaLigator software i, i = 1,2,…etc.) for each binding site as described 2+], hence yielding the apparent affinity constants. For the fitting the precise concentration of each protein sample stock was determined by amino acid analysis after acid hydrolysis .CaStandard biochemical techniques as SDS polyacrylamide gel electrophoresis and determination of protein content were done according to well established protocols in our laboratory 2+-loaded zGCAPs have an apparent molecular mass of 19–21 kDa as analysed by SDS polyacrylamide gel electrophoresis and the electrophoretic mobility became less in the absence of Ca2+ by adding EGTA. Interestingly, this Ca2+-shift differed among the tested zGCAPs as it was most prominent for zGCAP2 and zGCAP5 . So far, only for zGCAP4 we were able to investigate the myristoylated and non-myristoylated forms of the protein in more detail and found no significant differences in key properties of its Ca2+-sensor function All six zebrafish specific GCAP forms could be purified from bacterial lysates using anion exchange chromatography, ammonium sulfate precipitation and size exclusion chromatography. Although all isoforms could be purified from soluble cell fractions higher yields were obtained by purifying and renaturing zGCAP1, 2, 5 and 7 from inclusion bodies. In control experiments no differences were observed between proteins from soluble fractions or from inclusion bodies. All Cad zGCAP5 . Further50 value gives the particular zGCAP concentration that half-maximally activates GCs, the IC50 value gives the [Ca2+] at which the GC activity is halfmaximal and the x-fold activation is expressed as GCmax−GCmin divided by GCmin  for nearly all zGCAPs at a minimum. It constantly changed to higher intensity values at increasing [Ca2+]  mirrors the Ca2+-induced conformational change in zGCAP7 leading to a different hydrodynamic volume of the protein. Other zGCAP forms did not undergo a monomer-dimer equilibrium shift; instead they remained to a large extent in a monomeric state as this was seen with zGCAP7 in the presence of Ca2+  was obtained for all zGCAPs and one low affinity site was measured with zGCAP1, zGCAP2 and zGCAP5 (Kl≥5 µM). A particular case was observed with zGCAP7 that contained one high affinity and three medium affinity sites  up to a factor of 48 (zGCAP2).Direct binding of Caty sites . The resty sites . Transfon curves  allowed o zGCAPs . Among d2+-sensor system with different biochemical properties. Most significant are differences in the IC50 values  are still missing, but Ciluffo et al. suggested from their measurements on visible cones a mean dark value around 400 nM 2+] which is followed by the well understood light-triggered decrease of cytoplasmic [Ca2+] in photoreceptor cells via the operation of the Na+/Ca2+, K+-exchanger. This complex pattern of changes in intracellular [Ca2+] had previously been detected in cones from the zebrafish mutant nof2+-induced conformational changes and regulated membrane bound GC between 30 and 500 nM [Ca2+], but why do cone cells express more than one form of zGCAP?Direct measurements of cytoplasmic  than in other cone types, but so far a comparative analysis of changes in cytoplasmic [Ca2+] in different cone types is missing.Our data suggest that the different cone subtypes harbour combinations of zGCAPs that exhibit complementary properties. 2+], where these zGCAPs dynamically regulate GC, is expanded with IC50 values between 30 and 520 nM.In long single cones and double cones transcription of zGCAP3, zGCAP4 and zGCAP5 2+-affinity of each zGCAP were broadly consistent with the Ca2+-sensitive regulation of GC, since the apparent binding constants were in the nanomolar to lower micromolar range  for Ca2+ leading to a zGCAP/GC complex that has a higher Ca2+-affinity than the isolated protein. This form of intermolecular tuning of a Ca2+-sensor by its target has been previously observed with calmodulin and is an effective way to allow one small regulatory protein  to interact with a large variety of proteins in a cell 2+-affinities can be explained by free-energy coupling 2+ to calmodulin influences its affinity to a target in the same manner as the interaction of calmodulin with a target changes its affinity for Ca2+. The same behavior has been hypothesized for GCAP1/GC interaction 2+-sensitivity of zGCAPs might become even more complex, if we consider that the zebrafish retina expresses three sensory GCs, one being cone specific and two being expressed in rods and UV cones However, apparent Ca2+ and Trp fluorescence spectroscopy indicated that all zGCAPs exhibited a Ca2+-induced conformational change. Ca2+-dependent conformational changes belong to the key properties of Ca2+-sensors and are thought to represent the activating trigger step in a GCAP molecule that activates its associated target GC 2+] 2+, which clearly differs from the observation made with bovine GCAP isoforms 2+] (designated as phase I) correlate with amino acid side chain movements around Trp 21 and 51 and changes above 1 µM [Ca2+] (designated as phase II) with movements around Trp 94 2+ in bovine GCAP1 2+-dependent intensity change in Trp fluorescence for zGCAP1,2,3,4 and 5, although we observed in the gel shift assay that Mg2+ has an influence on the electrophoretic mobility indicating binding of Mg2+ to zGCAPs. Among all zGCAPs, zGCAP7 has a unique distribution of Trps in the amino acid sequence, present at positions 94, 164 and 178. Although a Trp is missing at position 51, we observed for zGCAP7 a Mg2+-effect below 1 µM free [Ca2+] (2+].Limited proteolysis in the presence and absence of Cae [Ca2+] . Therefo2+ and zGCAPs, of which each zGCAP form displayed a very specific detection mode for Ca2+. In photoreceptor cells cytoplasmic [Ca2+] is expected to be balanced at levels that finally depend on the illumination conditions 2+-relay model previously hypothesized for the function of GCAP1 and GCAP2 in bovine rods 2+-fluctuations in a cone cell are therefore adjusted by the Ca2+-homeostasis that is under control of the illumination conditions. Each range of cytoplasmic [Ca2+] would be optimal for a specific zGCAP to operate as an activator of GC. The differential fingerprint properties of zGCAPs fit into a step by step regulation of GC activity triggered by changing [Ca2+]. This zGCAP signaling system might be part of the molecular toolbox enabling the cones to respond to a large range of light intensities. The specific challenges of the zebrafish habitat, for instance, the dominance of short wavelengths in ambient background light, probably caused genome duplication under evolutionary pressure, which is now mirrored in the diversity of zGCAP expression (for an extensive discussion on this point see In summary, our data indicate the existence of a complex interplay between Ca