Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2Rγcnull mice . We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies.Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current  Overall survival at 5 years is ~30–40% in patients younger than 60 years, and <20% in patients over 60 years. Front-line chemotherapy is highly effective in ablating leukemic cells, but distant relapses are observed in the majority of patients, characterized by a refractory phase during which no other treatment has shown any efficacy thus far. Relapses are caused by malignant cell regrowth initiated by chemoresistant leukemic clones.3Acute myeloid leukemia (AML) is the most common adult acute leukemia and is characterized by clonal expansion of immature myeloblasts, initiating from rare leukemic stem or progenitor cells. In Europe and the USA, the incidence and mortality rates of AML are about 5 to 8/100.000 and 4 to 6/100.000 per year, respectively.cnull (NSG) mice.5 These highly immunodeficient mice have the advantages of a longer life span and higher levels of engraftment of human AML cells compared with other immunocompromised mouse strains such as SCID, NOD-SCID.7 However, this model is based on three-to-six month-long experiments with a large excess of cells and requires access to a biobank of diverse primary AML patient samples. Moreover, extensive characterization of this xenograft model with more commonly used and easily accessible AML cell lines has not been reported to date in the same setting to compare models in vivo. Herein, we report experimental conditions (bone marrow preconditioning and injected cell number) for a faster and easier preclinical mouse model of AML based on the xenotransplantation of a panel of six adult and childhood human AML cell lines, representing different FAB types, genetic variants and chromosomal abnormalities. In addition, we have also shown that our cell line-derived xenograft (CLDX as mirrored to patient-derived xenograft) models exhibited a broad range of overall mice survival, engraftment, tissue infiltration and aggressiveness based on their AML cell line characteristics.This unfavorable situation leads to a strong need for new therapeutic strategies, as well as relevant preclinical mouse models in which to test them. We have previously established a robust xenotransplantation model to study primary human AML biology and stem cell function based on the engraftment of primary AML samples into non-obese diabetic (NOD)/LtSz-severe combined immunodeficiency (SCID) IL2Rγ2. The cultured cells were split every 2–3 days and maintained in an exponential growth phase. All cell lines were annually re-ordered to DSMZ or ATCC stocks and periodically authenticated by morphologic inspection and mutational sequencing, and tested negative for Mycoplasma. The clinical and mutational features of our AML cell lines are described in Human AML cell lines U937, MV4-11, MOLM-14, HL-60  and KG1a  were maintained in minimum essential medium-α medium supplemented with 10% fetal bovine serum . All cell lines were grown in the presence of 100 units per ml of penicillin and 100 μg/ml of streptomycin, and were incubated at 37 °C with 5% COCD45-APC , CD33-PE (BD 555450), CD44-FITC (BD 555478), CD44-PE-Cy7 (BD 560533), CD45.1-PerCP-Cy5.5 (BD 560580), CD34-APC-Cy7 , Lin1-FITC (BD 340546), CD123-PE (BD 340545), CD45RA-Alexa Fluor 700 (BD 560673) and CD38-APC (BD 555362) fluorescent antibodies were used to analyze leukemic cells before and after injection into animals to determine phenotypic analysis of engrafted cells and percentage of leukemic cell engraftment. Absolute cell counts were determined with CountBright absolute counting beads (Invitrogen) following the manufacturer's recommendations.Animals were used in accordance with a protocol reviewed and approved by the Institutional Animal Care and User Ethical Committee of the UMS006 and Région Midi-Pyrénées . NSG mice were produced at the Genotoul Anexplo platform at Toulouse (France) using breeders obtained from The Charles River Laboratory. Mice were housed in sterile conditions using high-efficiency particulate arrestance filtered micro-isolators and fed with irradiated food and acidified water.Adult mice (6–8 weeks old) were sublethally irradiated with 250 cGy of total body irradiation or treated with 20 mg/kg busulfan  by intraperitoneal administration 24 h before injection of leukemic cells. Cultured AML cell lines were washed twice in phosphate-buffered saline (PBS) and cleared of aggregates and debris using a 0.2-mm cell filter, and suspended in PBS at a final concentration of 0.2–2 million cells per 200 μl of PBS per mouse for intravenous injection. Xenograft tumors were generated by injecting AML cells (in 200 μl of PBS) in the tail vein of NSG mice. Daily monitoring of mice for symptoms of disease  determined the time of killing for injected animals with signs of distress.Peripheral blood was obtained with retro-orbital bleeding. Fifty microliter of blood were collected in heparin-coated collection tube for analyzed with hematology counter .NSG mice were humanely killed in accordance with the Institutional Animal Care and User Ethical Committee of the UMS006 and Région Midi-Pyrénées Protocols. Bone marrow (mixed from tibias and femurs) and spleen were dissected, crushed in PBS and made into single cell suspensions for analysis by flow cytometry .P-values. Significance is represented as followed: *P<0.05, **P<0.01 and ***P<0.005.Mann–Whitney test was used to calculate final in vivo the five most commonly used AML cell lines with a range of molecular abnormalities, clinical, biological and immunophenotypical characteristics  required for the evaluation of therapeutic agents in routine and easy-to-use setting. The advantages of using well-established AML cell lines as compared with primary patient samples are the unlimited access to a large amount of human AML cells and a faster engraftment in immunodeficient mice. We analyzed eristics . MOLM-14+ cells is greatly enhanced if recipients receive a chemical conditioning regimen such as busulfan  or total body irradiation (up to 4 Gy).11 However, it is not clear whether conditioning improves the engraftment of human AML cell lines especially in the most recently developed NSG mouse model. We first performed a maximally tolerated dose study to define the best conditioning procedures  decreased the overall mice survival for all AML cell lines tested  depending on the AML cell line type  exhibited a broad range of mice survival, engraftment, tissue infiltration and aggressiveness. By increasing or decreasing the number of injected leukemic cells, we can modulate mice survival without changing the tissue distribution of leukemic cells and their engraftment in NSG mice. This work has also shown that AML cell lines kill mice in a manner similar to that as patients in clinics but different from the xenograft of primary AML patient specimens in NSG mice. Moreover, in contrast to patient-derived xenograft models, these CLDX models are a powerful, rapid and straightforward in vivo assay available to all leukemia research laboratories to perform preclinical studies to assess the in vivo efficacy of conventional or targeted therapies in AML.Here we provide necessary technical details and conditions that other laboratories may use to quickly and routinely set up


Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation  or activation  related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.The Ca Like other members of family C in the GPCR superfamily, the CaSR possesses a large extracellular domain (ECD) consisting of more than 600 amino acids 2+, the principal physiological agonist for CaSR, is thought to bind to the ECD of the dimeric form of the receptor resulting in activation of phospholipase C (PLC), with an attendant accumulation of inositol 1,4,5-triphosphate (IP3) followed by a resultant increase in the cytosolic calcium ([Ca2+]i) concentration The human calcium  cause familial hypocalciuric hypercalcemia (FHH) and neonatal severe primary hyperparathyroidism (NSHPT) 173 and Pro221, where mutations of the same residue can either activate or inactivate the CaSR, serve as a rich source of structure-function information.Mutations of the CaSR can perturb intracellular signaling events  and disrupt regulation of PTH secretion from the parathyroid chief cell and Ca2+-binding sites predicted in our previous studies 2+-binding during receptor activation as well as for regulating both the homotropic and heterotropic cooperativity of CaSR Interestingly, among thirty-four identified ECD missense mutations in patients with FHH, NSHPT and ADHH, 18 were located within 10 Å of one or more of the five Ca2+-binding site 1 are dynamically correlated with those of the other predicted calcium-binding sites. Mutations in Ca2+-binding site 1 could, therefore, affect both the homotropic and heterotropic cooperativity of the CaSR. These results led us to hypothesize that residues 173 and 221 are essential for the functional cooperativity orchestrated by calcium and L-Phe binding, and that related disease-associated mutations are the result of alteration or perturbation of the receptor's associated molecular connectivity.Our previous computational studies showed that movements of Ca2+]o. Furthermore, L173P and P221Q disrupt the strong correlated motions among the various calcium-binding sites as demonstrated by molecular dynamics (MD) simulations. L-Phe induced positive heterotropic cooperativity of the P221Q mutant, but had only a limited effect in potentiating the functional activity of L173P due to its restricted molecular dynamical features. These in vitro and in silico results provide important insights into how Ca2+ and L-Phe interact at their respective binding sites in the cleft of the VFTD, which are essential for the maintenance of calcium homeostasis that is required for normal physiological function.In the present studies, we show that the loss-of-function mutations, L173P and P221Q, but not the gain-of-function mutations, L173F and P221L, alter the cooperativity of the CaSR though analyzing changes in functional readouts in response to alterations in o was increased in a stepwise manner in the presence or absence of 5 mM L-Phe. The ratio of emission fluorescence intensity from both excitation wavelengths was then monitored as a function of [Ca2+]o and utilized as a parameter reflecting changes in [Ca2+]i All experiments were performed at room temperature. In addition to the use of the cell population assay as a means of calculating EC50 for Ca2+o or L-Phe, the signals from 30 to 60 single cells were recorded for each measurement in a single cell [Ca2+]i assay. In the latter assay, [Ca2+]i oscillations in individual cells were defined as three or more successive fluctuations in [Ca2+]i from the baseline after the initial peak.Measurement of  ×100. The EC50s of [Ca2+]o-dependent responses were calculated by fitting the [Ca2+]o concentration-response curves with the Hill equation.HEK293 cells were seeded in 24-well plates at 3×102 (0–20 mM) with or without L-Phe (5 mM) for 10 min. At the end of the [Ca2+]o stimulation, cells were lysed with RIPA lysis buffer . In total, 150 µg aliquots of lysate protein were loaded into either a 12.5% SDS-gel or a 4%–12.5% gradient gel for PAGE and analyzed by western blotting with an anti-phospho-p44/42 ERK polyclonal antibody  diluted (1∶2000). A chemiluminescent method (AP Conjugate Substrate Kit) was employed to detect the phospho-(p)44/42 proteins. Quantitative analysis of the results was performed using ImageJ software . The responses were normalized to the maximal effect observed with [Ca2+]o alone. The EC50 of [Ca2+]o-dependent responses was calculated by fitting the [Ca2+]o concentration-response curves with the Hill equation.Thirty-six hours post transfection of monolayers of HEK293 cells with CaSR or its mutants, cells were incubated in serum-free high glucose DMEM medium supplemented with 0.2% w/v BSA at 37°C overnight. On the following day, cells were first incubated with HBSS for 30 min, followed by stimulation with varying levels of CaCl2+-binding sites in the CaSR ECD were predicted using MetalFinder CLUSTALW was used to align the sequences of the human CaSR ECD (residues 25–530) and the mGluR1 ECD 2+-binding site, but no restraints were used during the actual simulation. A total of three MD simulations were carried out for 50 ns each on the apo-form and the ligand-loaded forms. During the simulations, an integration time step of 0.002 ps was used to solve the Newton's equation of motion. The long-range electrostatic interactions were calculated using the Particle Mesh Ewald method, MD simulation provides an approach complementary to experiments in live cells for understanding biomolecular structure, dynamics, and function. The initial coordinates for all the simulations were taken from a 2.20 Å resolution x-ray crystal structure with PDBID 1EWK The data are presented as means ± SE of the indicated number of experiments. One-way repeated measures analysis of variance (ANOVA), followed by the Dunnett post hoc test was performed using SPSS  for multiple comparisons to compare the differences between groups without adding L-Phe. Two-way ANOVA was used for comparison among different groups in the presence of L-Phe. A P value of < 0.05 was considered to indicate a statistically significant difference.173, Pro221) involved in the four disease-associated mutations studied here are located in the hinge region near the predicted calcium-binding site 1, which is located between lobe 1 and lobe 2. Meanwhile they are also adjacent (within 10 Å) to the reported L-Phe binding pocket formed by residues K47, L51, W70, T145, G146, S169, S170, I187, Y218, S272, H413 and R415, which can exert effects over a large region of the ECD through positive heterotropic cooperativity as shown in The two residues , the majority of the cells, 79% for L173F and 40% for P221L, started to oscillate at lower levels of [Ca2+]o, 2.0 mM and 2.5 mM, respectively.Analysis of the o  & 3. How2+]o  started to oscillate—15 mM for L173P and 5 mM for P221Q. For L173F and P221L, the frequencies were measured at 2.5 mM [Ca2+]o. The oscillation frequency for the two inactivating mutants did not differ from that of the WT (1.3±0.1 peaks/min) and were 1.3±0.1 peaks/min for L173P and 1.5±0.1 peaks/min for P221Q, but there were slight but significant increases for L173F (1.7±0.1 peaks/min) and P221L (1.6±0.1 peaks/min).WT CaSR-transfected cells exhibited a normal distribution of oscillation frequencies with an average frequency of 1.0∼1.5 peaks/min at 3.0 mM [Ca2+]o , Table 12+]i plateau  at 5.0 mM [Ca2+]o  or a decrease in Hill number for P221Q, accompanied by an increase of EC50 to 5.2±0.4 mM , which induces an increase in [Ca2+]i caused by the associated accumulation of inositol phosphates (IPs) 2+]o in cells transiently transfected with WT CaSR were assessed by measuring the accumulation of IP1 as an index of PLC activation 50 for the stimulation of IP1 accumulation was 2.9±0.3 mM, similar to the EC50 calculated from the [Ca2+]i responses (3.0±0.2). As with their Ca2+i responses, L173P and P221Q exhibited impaired [Ca2+]o–stimulated IP1 accumulation as reflected by the increases in their EC50s, 10.7±1.0 mM and 6.1±0.3 mM respectively, vs. 2.9±0.3 mM for the WT)  , Table 22+]o on mitogen-activated protein kinases (MAPK) (ERK1/2) activation. Exposure of WT CaSR-transfected HEK293 cells to increasing concentrations of [Ca2+]o in the range of 0.0-∼2.0 mM for 10 minutes had little effect on the phosphorylation of p44/42 ERK. Greater increases of [Ca2+]o resulted in the accumulation of p44/42 ERK, which exhibited a maximum response at 8.0 mM [Ca2+]o. Immunoblots of [Ca2+]o–induced increases in ERK activity were further quantified using ImageJ and plotted against [Ca2+]o. The data were then fitted using the Hill equation, giving an EC50 of around 3.0±0.1 mM, which is close to the result measured using intracellular calcium responses. The mutants L173P and P221Q also impaired the [Ca2+]o-triggered ERK activity of the CaSR. More than 8.0 mM [Ca2+]o was needed in order to induce the phosphorylation of ERK1/2 for both mutants , L-Phe lowered the threshold for the termination of [Ca2+]i oscillations, but barely shifted the frequency distribution of their starting points nor significantly increased their frequencies at 2.5 mM [Ca2+]o  and increasing the Hill coefficient to 5.0±0.7 mM (50 (3.7±0.3 mM) with an increase in Hill number (4.1±0.7 mM) for mutant P221Q as measured from the [Ca2+]o-evoked increase in [Ca2+]i. L-Phe did not left-shift the activation of the L173P mutant by [Ca2+]o to the same extent that it did with the other loss-of-function mutant, P221Q, potentially because of the proximity of this residue to the binding site for L-Phe, which might interfere with L-Phe binding. The disruption of the positive homotropic cooperativity induced by [Ca2+]o as a result of the Leu to Pro change, which was reflected as a biphasic concentration response curve, was also not corrected by the addition of L-Phe . In the case of the mutant P221Q, the EC50 was reduced from 6.1±0.3 mM to 4.5±0.4 mM in the presence of L-Phe (p<0.05). On the contrary, L-Phe failed to reduce the EC50 of L173P significantly (9.0±0.9 mM). The EC50s of the IP1 responses for the two receptors with activating mutations, L173F and P221L, were not altered by L-Phe either , but had little influence on the activity of mutant L173P  to L-Phe compared to WT and the other mutants . Since the influence of L-Phe might, to some extent, depend on the extracellular calcium concentration, the apparent affinities of L-Phe for the two loss-of-function mutants were further analyzed at levels of [Ca2+]o equal to their respective EC50s for [Ca2+]o, specifically 15.0 mM [Ca2+]o for L173P and 5.0 mM [Ca2+]o for P221Q. P221Q exhibited a reduced EC50 for the L-Phe response  at 5.0 mM [Ca2+]o compared with EC50 for L-Phe measured at 1.5 mM [Ca2+]o (EC50 = 4.5±0.4 mM), while L173P retained a similar sensitivity to L-Phe regardless of the extracellular calcium concentration   , Table 32+ in site 1 produces greater correlated motions among the Ca2+-binding sites compared with L-Phe alone in site 1 173 and Pro221 play pivotal roles in modulating the molecular connectivity between the binding sites for [Ca2+]o. In order to examine the possibility that the effects of inactivating mutations of the CaSR can be cancelled by those of activating mutations through molecular correlated motions, two double mutations, L173F/P221Q and L173P/P221L have been analyzed. We found that L173F/P221Q behaved like WT CaSR, thereby exhibiting a “cancelling” effect  separates out the protein motions into principal modes ranked according to their relative contributions ic level .2+ and L-Phe, the conformations sampled by the CaSR mutants are similar to that of the WT CaSR. However, in the L-Phe and Ca2+-loaded forms, the conformations of the CaSR mutants L173P and L173F are distinguishable from the other mutants and WT CaSR  and the holo forms (lower panel) onto the first three modes that accounted for the majority of the total fluctuations is shown in  WT CaSR . The res2+-binding site 1 are hypothesized to influence the functional cooperativity of the entire receptor. In vitro studies showed that the L173P and P221Q mutants produce a receptor with reduced positive homotropic cooperativity and impaired capability to sense [Ca2+]o, while the L173F and P221L mutants enhance the sensitivity of the CaSR to [Ca2+]o compared to the WT receptor, but barely change the cooperativity of the receptor. The results with the loss-of-function mutants indicate that Leu173 and Pro221 are important in maintaining the positive homotropic cooperativity among the receptor's calcium-binding sites. Conversely, the gain-of-function mutants show that these residues also are important for restraining the receptor from assuming its most active form . Thus unlike mutations of residues that cause only gain- or loss-of-function, Leu173 and Pro221 serve dual functions, perhaps reflecting their key locations in the cleft between the receptor's two lobes and their proximities to the binding sites for both [Ca2+]o and L-Phe.The CaSR hinge region is considered to be crucial for the sensing of agonists  2+]i oscillations have been postulated to be the result of complex responses from multiple signaling pathways. The [Ca2+]o-triggered [Ca2+]i oscillation pattern is believed to be modulated by the activity of phosphoinositide pathway 3 production 1 and the activity of ERK1/2, which will be discussed later, can be reflected in the oscillation patterns associated with the different disease-related mutations. Although most research has focused on intracellular calcium mobilization in HEK293 cells heterologously expressing the CaSR, CaSR-induced [Ca2+]i oscillations have also been found in cells with endogenously expressed CaSR, for instance in parathyroid cells 2+]i modulate not only the rate of parathyroid hormone (PTH) secretion, but also gene expression and other processes 2+]i oscillations is thus one of the most important signatures reflecting the state of CaSR activity. In the present study, we showed that the inactive mutants required higher [Ca2+]o to trigger intracellular [Ca2+]i oscillations compared to the WT CaSR, while the active mutants needed lower [Ca2+]o to initiate oscillations. The oscillation frequency of the inactive mutants remained similar to that of the WT CaSR, but higher [Ca2+]i oscillations frequencies were observed in HEK293 cells transfected with gain-of-function mutations. As [Ca2+]o is increased, the [Ca2+]i oscillations cease and reach a plateau in CaSR-transfected cells as the desensitization process occurs and/or there is depletion of [Ca2+]i stores. Although the loss-of-function mutation L173P still oscillated at 30.0 mM [Ca2+]o, considering the elevated and broad range of its sensitivity to [Ca2+]o, it is possible that a plateau would be reached at even higher levels of [Ca2+]o.i oscillations that at least partially involve activation of the Gαq/11 pathway. The changes in the accumulation of IP1 upon stimulation with various concentrations of [Ca2+]o correlated well with [Ca2+]i changes. Secondly, high [Ca2+]o-evoked increases in ERK1/2 activities of WT-CaSR and its four mutants were in accordance with the accumulation of IP1. Stimulation of the ERK1/2 activity in CaSR-transfected HEK293 cells has been reported to involve PKC-mediated as well as a PTX-sensitive, tyrosine kinase-dependent pathways Our studies of CaSR-mediated accumulation IP2+]o triggered [Ca2+]i oscillation patterns in all of the mutants as measured in individual cellular responses . The alterations in the oscillation patterns after the introduction of the allosteric activator could be the result of the activation of signaling downstream of Gα12/13 or a combined effect with the positive heterotropic cooperativity between the multiple Ca2+ binding sites induced by L-Phe. The interaction between CaSR and L-Phe has been reported to modulate the Gα12/13-RhoA pathway 2+]o-induced alterations in intracellular signaling pathways through Gq/11 pathway as reflected by the IP1 accumulation assay.In the current study, we showed that L-Phe could affect one or two of the three parameters depicting the [Ca2+]o but also by the positive allosteric modulator, L-Phe 2+]o-triggered [Ca2+]i and IP1 responses for these mutants, which may explain, in part, the dramatic changes in correlated motions from the computational simulation results using molecular dynamics. As a signaling messenger further downstream than either IP1 or [Ca2+]i, the phosphorylation of ERK1/2 may not merely reflect activation of the Gq/11 pathway, but could be contributed to by other G-proteins . In agreement with the in silico results, L-Phe exhibited less effect on the regulation of the ERK1/2 pathway in mutant L173P. The predicted L-Phe-binding site has been reported to be located near the hinge region between lobe 1 and lobe 2 and to involve residues S169A/S170A/S171A 2+]o. Thus, the hetero-cooperativity between the allosteric modulator and calcium binding sites could be disturbed, resulting in the lack of a left-shifted concentration-response curve in response to [L-Phe].The present study also confirms that the ERK signaling pathway in CaSR-transfected HEK293 cells can be modulated not only by o by a heterotropic positive cooperative mechanism. Mutations close to the hinge region such as those at Leu173 and Pro221 affect the overall dynamic correlated motions of the respective mutant receptors and the Ca2+-induced homotropic cooperativity, which could further influence the conformation of the receptor. The inactivating mutation L173P impairs the molecular connectivity in the WT CaSR and cannot be rescued by L-Phe. On the other hand, the inactivating mutation P221Q weakens the connectivity but can be partially rescued by binding Phe with a resultant activity similar to the basal activity of the WT CaSR. In contrast, the gain-of-function mutants L173F and P221L may already have been converted to the active state with dynamic properties similar to that of Ca2+- loaded WT CaSR. They can be further activated upon binding extracellular Ca2+ to reach a state close to the “fully active” state (Phe + [Ca2+]o in the WT) that is insensitive to further activation by L-Phe achieving an apparent “ceiling” represented by the maximal achievable activity of the CaSR.Based on all of the in silico simulations provide a novel method to predict mutational effects on CaSR will ultimately require the kind of structural detail available for the mGluRs, whose structures have been solved. Here, the model structure does not take into consideration the possible interactions between the ECD and the extracellular loops and/or transmembrane domain as well as the intracellular domains. Thus, caution is merited in over-interpreting the in silico results. Nevertheless, the predicted structural motions may help to identify the mutations or ligands that produce increased or diminished coupling among specific structural elements within the CaSR ECD. While this type of analysis is limited at the moment to the environment of the orthosteric site, this strategy could facilitate drug design for modulating the functions of disease-related mutations affecting the CaSR, as well as other members of the family C GPCRs.The results from PCA may also provide an explanation as to why L-Phe interacts preferably with the P221Q mutant. L-Phe could not effectively shift the inactive population of conformational ensembles of L173P to an active ensemble of conformations as it did with the other inactivating mutant and WT. However, whether the 2+]o on different mutant CaSRs also suggests that ligand selectivity at the orthosteric site and receptor activity at non-orthosteric sites could both contribute to the function of the receptor. This feature of the adjacent orthosteric binding sites and allosteric regulatory sites that cooperatively regulate the molecular connectivity may be shared by other members of the family C GPCRs 2+-sensing receptor, [Ca2+]o regulates 14 of the other members of the family C G protein-coupled receptors (GPCRs), including the metabotropic glutamate receptors (mGluR), γ-aminobutyric acid GABAB receptors and receptors for pheromones, amino acids and sweet substances The distinctive combinatorial effects of L-Phe and [Ca2+]i oscillations and the [Ca2+]o- and L-Phe triggered downstream signaling changes, functional positive cooperativity of CaSR was revealed to be disrupted in the identified FHH-associated CaSR mutations, L173P and P221Q, but not in the ADHH related mutations, L173F and P221L. The addition of L-Phe rescued the function of P221Q by inducing heterotropic positive cooperativity. The distinctive correlated motions of the gain-of-function mutations, on the one hand, and the loss-of-function mutations, on the other, and the potential utility revealed by the molecular dynamic simulation in providing insights into the mechanism for the disease-associated functional alterations in the receptor may also apply to other GPCR proteins.In conclusion, through analysis of [CaFigure S1Functional studies of the CaSR double mutations in individual HEK293 cells. The panels show representative oscillation patterns from single cells. HEK-293 cells transfected with L173F/P221Q or L173P/P221L were loaded with Fura-2 AM for 15 min. [Ca2+]i was assessed by monitoring emission at 510 nm with excitation alternately at 340 or 380 nm as described in 2+-free Ringer's buffer , followed by stepwise increases in [Ca2+]o until [Ca2+]i reached a plateau (up to 30 mM [Ca2+]o). a. Cells were transfected with pEGFP-N1-CaSR L173F/P221Q. b. Cells were transfected with pEGFP-N1-CaSR L173P/P221L.(DOCX)Click here for additional data file.


SR) function in the absence and presence of β‐adrenoceptor stimulation. ZD7288 [to block I(f)] caused a substantial reduction (222 ± 13 bpm) in beating rate from 431 ± 14 to 209 ± 14 bpm, ryanodine alone (to block SR Ca2+ release) reduced beating rate by 105 ± 11 bpm, with subsequent addition of ZD7288 further reducing rate by 57 ± 9 bpm. Cyclopiazonic acid (CPA) alone (to inhibit Ca2+ reuptake by the SR) reduced beating rate by 148 ± 13 bpm with subsequent addition of ZD7288 further reducing rate by 79 ± 12 bpm. In additional experiments measuring Ca2+ transients in the SA node region using Rhod‐2, effects of ivabradine and ZD7288 on rate were again substantially reduced after CPA. Effects of CPA alone on rate developed much more slowly than effects on Ca2+ transient amplitude. ZD7288, ivabradine, and CPA reduced the slope and maximum response of the log(concentration)–response curves for effects of isoprenaline on beating rate. Very little response to isoprenaline remained after treatment with CPA followed by ZD7288. Similar changes in isoprenaline log(concentration)–response curves were seen in guinea pig preparations. These observations are consistent with a role for Ca2+ released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR‐derived Ca2+ to effects of β‐adrenoceptor stimulation on beating rate that are independent of I(f).The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac “funny” (f) channels, I(f), and/or suppression of sarcoplasmic reticulum ( For experiments in which cytosolic Ca2+ was to be measured using Rhod‐2, the preparations were subjected to additional dissection to reveal the translucent region in the right atrium adjacent to the crista terminalis. This translucent region contains the site of origin of pacemaker activity (see below) and was visualized with an EMCCD camera in experiments in which the amplitude and timing of Ca2+ transients were measured.The protein ion channel that carries I(f) is made up of HCN (Hyperpolarization activated Cyclic Nucleotide regulated) subunits 1–4, with HCN4 probably playing the most important role in the heart. Genetic work shows that suppression of HCN4 slows but does not abolish spontaneous activity  base containing oxygenated Physiological Salt Solution (PSS) at room temperature. The ventricles were dissected away leaving intact isolated atrial preparation.All procedures involving animals were approved by the Oxford University ethics board in accordance with the Home Office (UK) Code of Practice and Animals (Scientific Procedures) Act 1986 guidelines. Schedule 1 was performed on male CD1 mice and guinea pig by initial stunning followed by cervical dislocation. The heart was rapidly excised and placed in a chamber with a SylgardFollowing the removal of ventricles, nonabsorbable braded silk‐waxed suture  was used to carefully tie a basic surgical knot around the lateral wings of the two atria. The preparation was then transferred to a 20 mL organ bath containing 10 mL oxygenated PSS, warmed by a water jacket to 37°C. The atria were mounted vertically such that the left atrium was attached to a metal hook positioned at the bottom of the organ bath and the right atrium was attached to an isometric force transducer . The beating rate was calculated from the interval between contractions using Chart 5 software. The beating rate was also calculated using an extracellular electrode placed close to the SAN region to record a compound action potential signal. The beating rate was calculated from the interval between individual extracellular voltage signals associated with compound action potentials using Chart 5 software. Thirty‐minute incubation of the tissue with SR inhibitors, MDL‐12330A, ivabradine, and ZD7288 was required to reach a reduced plateau steady beating rate. The rate (beats per minute) was measured from beats sampled over a period of approximately 20 sec. The preparation was allowed to stabilize for 45 min before an experiment was carried out.2+ probe) and incubated in PSS for 30 min with 10 μmol/L blebbistatin, followed by 45 min incubation with 4 μmol/L Rhod‐2, AM. The solution was then replaced with PSS containing 10 μmol/L blebbistatin; the preparation was allowed to stabilize for 10 min before experimentation. The location of the leading pacemaker site was observed as a stable fluorescence signal resulting from excitation of Rhod‐2, originating from right atrium site near the superior vena cava where the SAN is anatomically defined. Upon detection of the leading pacemaker site we selected region of interest corresponding to an area of 375 by 375 μm from its central core to record Ca2+ transients. Under the conditions of our experiments the leading pacemaker site remained stable during application of drugs. Thirty‐minute incubation of the tissue with SR inhibitors and ZD7288 was required to reach a reduced plateau steady beating rate. The recording was sampled over 10 sec at a frame rate of 3000 frame/sec. The above procedures did not interfere with the spontaneous rhythm or cause any irregularities of the rhythm. In particular, little or no change in heart rate was detected by a surface electrode to record extracellular compound action potentials following application of blebbistatin and Rhod 2 .Vessels and connective tissue from the external surface of the isolated atrial preparation surrounding the superior vena cava were carefully removed without interfering with the posterior wall of the node. The preparation was then transferred to a 20‐mL chamber (with Sylgard base) containing 10 mL of oxygenated PSS. Next the atrial preparation was pinned to the Sylgard base and positioned so that the left atrium was on the right with the superior vena cava at the top. The right atrium was opened in order to expose the sinoatrial node area by making incision from the bottom of the right atrium toward the superior vena cava. This in turn exposed crista terminalis bands from the left‐hand side of the right atrium. Anatomically the sinoatrial node region differs from atrial tissue by being translucent and positioned close to the crista terminalis and away from the interatrial septum. The exposed beating atrial preparations were then kept at 34°C (to reduce the extrusion of the Ca2+ sensitive dye (Rhod‐2 AM). Emission filter, ET585/40 nm, was used for the collection of emitted fluorescent . An EMCCD camera with 128 × 128 pixels  was used for optical recordings. The sinoatrial node preparation was exposed to light only during image acquisition. The acquired fluorescent signal was visualized and quantified using custom software kindly provided by Gil Bub . Ca2+ transients were analyzed using the pCLAMP software.For illumination, we used four green LEDs with band‐pass filters, 560/50 nm, to excite the Ca3, 5.4 KCl, 1.2 NaH2PO4, 1.0 MgCl2, 5.5 glucose, 1.8 CaCl2, and the solution was aerated with 95% O2/5% CO2 to maintain a pH of 7.4. ZD7288, ryanodine, MDL12330A, and ivabradine were from Tocris Inc. Rhod‐2, AM, was from Life Sciences Technologies. All other chemicals were from Sigma Co. Except for isoprenaline and ryanodine, which were dissolved in distilled water, all other chemicals were dissolved in dimethyl sulfoxide (DMSO).Physiological salt solution (PSS) was prepared daily and contained (mmol/L) 125.0 NaCl, 25.0 NaHCOFor ZD7288, ivabradine, ryanodine, and MDL12330A an exposure time of at least 30 min was allowed, since it was found that there was a slow decline of spontaneous rate to a plateau steady state within this period. Spontaneous beating was generally regular during exposure to drugs, and any periods of irregularity were avoided for measurements of spontaneous rate.n = number of experiments. GraphPad prism (version 5.0) software was used to perform statistical analysis including Student's t‐test and two‐way analysis of variance (ANOVA), Bonferroni posttest .Results are expressed as means ± SEM, μmol/L) to investigate the effect of I(f) inhibition on spontaneously beating mouse atrial preparations (organ bath experiments in the absence of Ca2+ probe). The tissue was incubated with ZD7288 for 30 min, and during this time the beating rate reached a new lower steady state. Inhibition of I(f) with 1 μmol/L ZD7288 caused an approximate halving of the spontaneous beating rate. The spontaneous beating rate was reduced by 52 ± 5%, a reduction of 222 ± 13 bpm from a resting beating rate of 431 ± 14 bpm to 209 ± 14 bpm  or cyclopiazonic acid . Thirty‐minute incubation of the tissue with SR inhibitors was required to reach a reduced plateau steady beating rate. In the case of 30 μmol/L ryanodine, the spontaneous beating rate was reduced by 25 ± 6%, a reduction of 105 ± 11 bpm from a resting beating rate of 425 ± 18 bpm to 320 ± 17 bpm . With 100 μmol/L CPA, the spontaneous beating rate was reduced by 36 ± 4%, a reduction of 148 ± 13 bpm from 413 ± 19 bpm to 265 ± 14 bpm .To evaluate the contribution of Ca2+ release from SR (either with ryanodine or CPA). Compared with the 222 ± 13 bpm slowing reported above, the effects of ZD7288 on spontaneous beating rate after ryanodine or CPA appeared to be less than in the absence of drugs to suppress SR function: after ryanodine the change in beating rate was 57 ± 9 bpm  and after CPA the change in beating rate was 79 ± 12 bpm . Thirty‐minute incubation of a tissue with ZD7288 was performed after the 30‐min preincubation with SR inhibitor to reach a further reduced plateau steady‐state beating rate.We then investigated the effect of ZD7288 in slowing the spontaneous beating rate, but with initial suppression of Ca2+ that affects Ca2+‐sensitive adenylyl cyclases  on spontaneous rate are less when SR function is suppressed, perhaps because this leads to changes in cytosolic Ca2+) can be inhibited by MDL12330A . Exposure to MDL12330A reduced the basal beating rate from 443 ± 24 bpm to 131 ± 20 bpm  . It therefore appears that the effect of ZD7288 in slowing the spontaneous beating rate after MDL12330A was substantially reduced or abolished  relative to the reduction in rate observed in the absence of MDL12330A (approximately 200 bpm as reported above).Adenylyl cyclases (including those stimulated by cytosolic Caμmol/L), and is blockade of I(f) complete at this concentration? The selectivity of ZD7288 I(f) inhibition is likely to be high at 1 μmol/L since BoSmith et al. (2+ and K+ currents. We explored whether the blockade of I(f) was complete by exposing the tissue to additional drugs to test for further reductions in beating rate, as might occur if there were significant residual I(f) in the presence of 1 μmol/L ZD7288. Ivabradine is reported to be a selective I(f) blocker with little or no effect on other currents at 3 μmol/L . Ivabradine alone (3 μmol/L) showed a similar effect on spontaneous beating rate, causing a decrease from 378 ± 17 bpm to 159 ± 21 bpm, a reduction of 219 ± 20 bpm, 58 ± 5%, P < 0.05, n = 10. When 3 μmol/L ivabradine was added in the presence of 1 μmol/L ZD7288, there was little or no further change in beating rate . Experiments were also carried out at higher ZD7288 concentrations , while increasing the concentration to 3 μmol/L further reduced the beating rate by 46 ± 9 bpm  and subsequently increasing the concentration to 10 μmol/L caused a small additional reduction of 2 ± 6 bpm . Taken together, these observations are consistent with the view that blockade of I(f) by ZD7288 is near complete at a concentration of 1 μmol/L (see Two questions might be raised concerning the use of ZD7288 to block I(f): how selective is ZD7288 at the concentration used . Blebbistatin (10 μmol/L) was added to suppress contraction and avoid contraction artifacts in the optical signal for Ca2+. In order to assess whether blebbistatin or Rhod‐2 had any effect on basal beating rate, an external electrode was used placed close to the SAN region to record the extracellular compound action potential signal. Forty‐minute incubation with blebbistatin (10 μmol/L) together with Rhod‐2, AM (4 μmol/L) was associated with a small fall in basal beating rate , but this reduction in basal beating rate was not statistically different from that observed in the absence of Rhod‐2 and blebbistatin  appeared to be less when SR function was suppressed. This apparent reduction in effect could occur if the activity of the Caod‐2, AM  μmol/L w2+ handling . During this period the amplitude of the Ca2+ fluorescence transient fell by 36 ± 5%, P < 0.05, n = 6, presumably as a consequence of extrusion of the Ca2+ probe, and/or possible dye photobleaching  was not significantly different from the reduction observed in the absence of drugs , presumably as a consequence of the suppression of SR function by inhibition of Ca2+ uptake by SERCA  and the data were well fitted by an exponential decay to a new steady rate of 129 ± 8 bpm with a time constant of 9.0 ± 1.8 min . It seems possible that these additional effects might develop more slowly, perhaps because it takes time for the change in Ca2+ levels to be sensed and/or for the proposed reduction in activity of Ca2+‐stimulated adenylyl cyclases to lead to slow changes in cAMP levels that are at least in part the result of Ca2+‐dependent reduction of this enzyme activity.At least some of the effect of CPA on beating rate presumably results from a reduction in sodium–calcium exchange current associated with the reduced CaCPA Fig. C: there P < 0.05, n = 6, Fig. P < 0.05, n = 6, at 70‐min period Fig. P < 0.05, n = 9, after 40 min). The observed reduced effect of ivabradine/ZD7288 under these conditions resembles the effects reported above  was blocked or SR function was suppressed. For these experiments, we returned to the simple organ bath preparation without Rhod‐2 or blebbistatin. In the absence of other compounds, isoprenaline caused a concentration‐dependent increase in beating rate with a maximum increase of 303 ± 10 bpm at 100 nmol/L. Several series of experiments were carried out with a variety of drug combinations. The initial aim was to block I(f) using 1 μmol/L ZD7288 (30‐min preincubation during which the beating rate declined to a steady‐state plateau); the presence of ZD7288 reduced the isoprenaline‐induced increase in beating rate at each concentration, and the reductions were statistically significant . The effects of a combination of 1 μmol/L ZD7288 and 3 μmol/L ivabradine  were similar those of 1 μmol/L ZD7288 alone. However, it was clear that substantial effects of isoprenaline on beating rate remained even in the presence of I(f) blockade that was thought to be close to complete with the combination of I(f) blockers  also depressed the log (concentration) – response to isoprenaline . The effects of these drugs that suppress SR function to cause a depression of the slope and maximum of the log(concentration)–response to isoprenaline were slightly greater than the effects of blockade of I(f) alone , there was a substantial further depression of the log(concentration)–response curve to isoprenaline relative to either CPA or ZD7288 when used alone. The change in beating rate at the maximum concentration of 100 nmol/L was substantially reduced to 51 ± 2 bpm, n = 10  and CPA to depress SR function were used together  alone (preincubating for 30 min) again depressed the log(concentration)–response curve to isoprenaline, so that the change in beating rate was less at all concentrations studied . The effects of a combination of 1 μmol/L ZD7288 and 3 μmol/L ivabradine (preincubating for 30 min to reach a steady‐state reduction) were similar those of 1 μmol/L ZD7288 alone . However, substantial increases in spontaneous beating rate in response to isoprenaline remained in the presence of these drugs to block I(f). At the highest concentration tested, 100 nmol/L, isoprenaline increased the beating rate by 85 ± 16 bpm in the presence of 1 μmol/L ZD7288, and by 88 ± 15 bpm in the presence of 1 μmol/L ZD7288 followed by 3 μmol/L ivabradine, compared with the control increase of 132 ± 21 bpm. The difference between the curves produced in the presence of 1 μmol/L ZD7288 compared to 1 μmol/L ZD7288 followed by 3 μmol/L ivabradine was not statistically significant. CPA  caused a greater depression of the maximum effect of isoprenaline at 100 nmol/L by 57 ± 9 bpm  than was observed with ZD7288 with or without ivabradine. Finally, the maximum effect of isoprenaline at 100 nmol/L in the presence of 1 μmol/L ZD7288 and 30 μmol/L CPA (with each drug being incubated for 30 min) was substantially suppressed to 8 ± 2 bpm compared to the control of 132 ± 21 bmp , and conclude that there are effects on Ca2+ cycling under the conditions of their experiments that contribute to the slowing of spontaneous rate. In particular, ivabradine was found to reduce SR Ca2+ load. This was tested with a rapid caffeine “spritz” and 3 μmol/L ivabradine was reported to reduce the amplitude of the caffeine‐induced Ca2+ transient by 13% . An indirect effect of a bradycardic agent on SR Ca2+ is not surprising as discussed above (since even when spontaneous rate is slowed by a completely selective I(f) blocker the fall in heart rate will reduce the number of action potentials per unit time that permit Ca2+ influx via voltage‐gated Ca2+ channels, and this reduction in Ca2+ influx would be expected to cause diminished Ca2+ uptake by the SR). Although an effect of this kind is expected, it appears that under the conditions of our experiments any effect was small  for our observations using the 3 μmol/L ivabradine and 1 μmol/L ZD7288 combination in mouse intact SA preparations, since there was little or no difference in the decline of the amplitude of Ca2+ transients in the absence and presence of these drugs over a 30‐min exposure period, even though these drugs caused an approximate halving of spontaneous rate. Future experiments will be required to investigate whether species differences  might account for the apparent discrepancy between our observations and those of Yaniv et al.  and SR‐dependent mechanisms to the β‐adrenoceptor pathways . Work with the canine SA node using fluorescent probes for Ca2+ also led to the conclusion that both I(f) and SR‐dependent mechanisms contribute to pacemaker activity  blockers, ZD7822 or ivabradine. This is unlikely to result from incomplete blockade of I(f) by these drugs at the concentrations used as discussed later. The effects of ZD7822 in combination with CPA profoundly suppress changes in spontaneous beating rate caused by μmol/L  reduction in beating rate and the degree of block caused by this drug is expected to be at least as high as that for ZD7288  blockade is technically difficult particularly with agents such as ivabradine that are thought to be “use dependent” and which take a long time to reach a steady‐state effect . “Rundown” of currents can also be a problem over long time periods. The first paper on ZD7288 quotes 74 ± 4% blockade at 1 D), where [D] is the drug concentration and KD is the dissociation equilibrium constant for a bimolecular binding reaction of the drug to the site/channel, then 75% block would be expected to occur when the concentration of each drug is three times greater than KD under the conditions of our experiments . Following the analysis of Jarvis and Thompson , the two drugs together would be predicted to leave a residual conductance of unblocked channels of 0.25 × 0.25 = 0.0625 of the total conductance or close to 94% blockade. If either of these estimates for blockade were underestimates , the estimates for combined block are even greater . In other words, the combination of 1 μmol/L ZD7288 and 3 μmol/L ivabradine may approach near complete blockade of I(f). The observation that there was little or no difference between the effects observed following 1 μmol/L ZD7288, 3 μmol/L ivabradine, or a combination of these two drugs may be rationalized by the proposal that 1 μmol/L ZD7288 and 3 μmol/L ivabradine each cause almost complete blockade of I(f) under these conditions, and that there is little further effect when the two drugs are combined. This view is also supported by the modest changes in spontaneous rate observed under these conditions when the concentration of ZD7288 was increased from 1 to 3 and then 10 μmol/L. The rate changes that we see in mouse when we believe little or no I(f) remains unblocked are remarkably similar to those observed by Mesirca et al.  channel, and that each drug separately occupies 75% of these sites under the conditions of our experiments .If these arguments are correct, their implications for pacemaker mechanisms underlying resting beating rate (in the absence of autonomic transmitters or hormones) can be considered. If very little I(f) remains in the presence of ivabradine and ZD7288, the observation that in mice the beating rate is reduced by approximately 50% by these drugs may indicate that residual mechanisms other than I(f) play a major role under these conditions. Such additional mechanisms could include both “membrane clock” mechanisms other than I(f), and “calcium clock” mechanisms. The membrane clock mechanisms include the importance of deactivating voltage‐gated K channels (together with an absence of a “stabilizing” resting K channel activity), sustained inward current, voltage‐gated Ca2+ release is suppressed could arise if SR‐derived Ca2+ were to activate Ca2+‐stimulated adenylyl cyclases such as AC1 or AC8, since this would lead to reduced levels of cAMP modulating I(f). Evidence supporting the contribution of such a pathway is provided by Mattick et al.  current was activated at potentials approximately 10 mV more depolarized than when HCN2 expression was coupled with AC6 or GFP  blockers on resting beating rates were less (measured as both % and bpm changes) in the presence than in the absence of ryanodine or CPA is consistent with a reduced contribution of I(f) under these conditions. Such reduced contribution of I(f) when SR Cak et al.  and Younk et al. . Functioμmol/L and 30 μmol/L, and it seems possible that nonspecific effects might occur, particularly at the higher concentration, but the observations with 30 μmol/L were remarkably similar to those at 100 μmol/L, consistent with a maximal drug actions. It is our experience that concentrations of blocking agents required for intact multicellular atrial preparations frequently appear to be higher than those required in isolated myocytes, perhaps because of barriers to permeation or other impediments to tissue penetration  to the positive chronotropic effect, isoprenaline will have effects via a direct G‐protein‐mediated stimulation of adenylyl cyclases, and possible additional effects that could arise as a consequence of an increased amplitude of the Ca2+ transient  would be expected to be apparent on this time scale. In contrast to the rapid effect of CPA on the amplitude of Ca2+ transients, it was observed that the full effect of CPA on spontaneous rate  took much longer to develop over a period of approximately 30 min. These observations are consistent with a slow development of effects of changed Ca2+ transients on Ca2+‐stimulated enzymes including AC1 and AC8. The slow development of effect could arise from a slow readjustment of cAMP levels as a consequence of changes in enzyme activity. The magnitude of I(f), and consequently the influence of this current on spontaneous rate, would be expected to closely follow the reduced levels of cAMP, but additional effects as a consequence of altered PKA function influencing other ion channels would also be predicted. It therefore seems likely that suppression of SR function will have effects on a number of pathways involved in the positive chronotropic response that are additional to any effects of I(f), as discussed in more detail below.Some of the effects of CPA to suppress the log(concentration)–response curve to isoprenaline and reduce the maximum chronotropic response might be attributable to a reduced I(f) as a consequence of the reduced amplitude of the Caβ‐adrenoceptor stimulation remained. From the arguments above concerning the effectiveness of these drugs it seems unlikely that the continuing effects of β‐adrenoceptor stimulation can be explained by effects on a large component of I(f) that remains unblocked. An additional experimental observation that supports the contention that the remaining effect of isoprenaline in the presence of the ZD7288 and ivabradine combination is not primarily a consequence of effects of isoprenaline on residual I(f) is the observation that so little of the effect of isoprenaline remained when the ZD7288 and ivabradine combination was supplemented with CPA.When I(f) was blocked by ZD7288 and ivabradine in various combinations, it appeared that substantial increases in beating rate in response to 2+, but do not involve this current. Targets for β‐adrenoceptor stimulatory mechanisms that could involve Ca2+‐stimulated adenylyl cyclases but not I(f) include pathways that are influenced by PKA following changes in cAMP levels. These include voltage‐gated Ca2+ channels, NCX , phospholamban/SERCA, voltage‐gated K channels, and sustained inward current channels  are best explained by effects of CPA that depend on SR Caβ‐adrenoceptor stimulation. Some of the effects of SR‐released Ca2+ can influence I(f) through activation of Ca2+‐stimulated AC1 and/or AC8, but there appear to be substantial additional SR‐dependent effects that are unrelated to I(f).In summary, our results are consistent with contributions from both I(f) and SR‐dependent mechanisms to pacemaker activity at rest and during None declared.


Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca2+ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca2+‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca2+ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca HL‐1 cells were seeded in fibronectin/gelatin‐precoated dishes as described previously. Cells were plated at a density of 5 × 105 cells/well in 6‐well culture plates. At 24 hrs after cell seeding, we added colchicine  HL‐1 cells derived from mouse atrial cardiac muscle cells  were cultured in a humidified atmosphere of 5% CO2+ (10 μmol/l) fluo‐3/acetoxymethyl (AM) ester for 30 min. at room temperature. Excess extracellular dye was removed by changing the bath solution, and intracellular hydrolysis of fluo‐3/AM occurred at 35 ± 1°C after 30 min. Fluo‐3 fluorescence was excited with a 488‐nm line of an argon ion laser. The emission was recorded at >515 nm. Cells were repetitively scanned at 3‐ms intervals for a total duration of 6 sec. Fluorescence imaging was performed with a laser scanning confocal microscope  and an inverted microscope . The fluorescent signals were corrected for variations in the dye concentration by normalizing the fluorescence (F) against the baseline fluorescence (F0) to obtain reliable information about intracellular (cytoplasm) Ca2+ ([Ca2+]i) transient changes from baseline values (F/F0) and to exclude variations in the fluorescence intensity with different volumes of injected dye. [Ca2+]i transients and peak systolic and diastolic [Ca2+]i values were measured during 2‐Hz field stimulation with 10‐ms twice‐threshold strength square‐wave pulses.As described previously 2+ transients with repeated pulses from −40 to 0 mV (1 Hz for 5 sec.), the total amount of charge crossing the membrane sarcoplasmic reticulum (SR) Ca2+ content (Ccaff) was estimated by integrating the sodium (Na+)–Ca2+ exchanger (NCX) current rapid application of 20 mM caffeine during rest with the membrane potential clamped to −40 mV to cause SR Ca2+ release 2+ content (expressed as mM of cytosol) was determined by use of the equation: SR Ca2+ content = [(1 + 0.12)(Ccaff/F × 1000)]/(Cm × 8.31 × 8.44), where Cm = membrane capacitance; F = Faraday's number; cell surface to volume ratio = 8.44 pF/pL After achieving steady‐state CaCa‐L) to avoid decay of ion channel activity over time. A small hyperpolarizing step from a holding potential of −50 mV to a test potential of −55 mV for 80 ms was delivered at the beginning of each experiment. The area under the capacitative current was divided by the applied voltage step to obtain the whole‐cell capacitance. The series resistance (Rs) was compensated by 60~80%. APs were elicited from isolated cardiomyocytes without spontaneous activity at a driven rate of 1 Hz for 20 beats. The resting membrane potential (RMP) was measured during the period between the last repolarization and the onset of the subsequent AP. The AP amplitude (APA) was obtained from the RMP to the peak of AP depolarization. AP durations (APDs) at 90%, 50% and 20% repolarization were, respectively, measured as the APD90, APD50 and APD20. Micropipettes were filled with a solution containing (in mM) KCl 20, K aspartate 110, MgCl2 1, MgATP 5, HEPES 10, EGTA 0.5, LiGTP 0.1 and Na2 phosphocreatine 5 (pH 7.2 with KOH) for the AP and potassium currents; containing (in mM) CsCl 130, MgCl2 1, MgATP 5, HEPES 10, EGTA 10, NaGTP 0.1 and Na2 phosphocreatine 5 (pH 7.2 with CsOH) for the L‐type Ca2+ current (ICa‐L); containing (in mM) NaCl 20, CsCl 110, MgCl2 0.4, CaCl2 1.75, tetraethylammonium 20, 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) 5, glucose 5, MgATP 5 and HEPES 10 (pH of 7.25) for the NCX current; and containing (in mM) potassium gluconate 144, MgCl2 1.15, EGTA 5, HEPES 10 and CaCl2 4.2 (pH 7.25 with KOH) for the apamin‐sensitive small‐conductance Ca2+‐activated K+ current (IKAS).A whole‐cell patch clamp was used for single isolated cardiomyocytes with an Axopatch 1D amplifier  at 35 ± 1°C Ca‐L was recorded using 300‐ms pulses from a holding potential of −50 mV to inactivate the T‐type Ca2+ current The I2 from that of the control. The recording protocol consisted of 300‐ms pulses ranging from −100 to +100 mV from a holding potential of −40 mV at a frequency of 0.1 Hz. The external solution for measuring the NCX contained (in mM): NaCl 140, CaCl2 2, MgCl2 1, HEPES 5 and glucose 10 (pH of 7.4). It was supplemented with strophanthidin (10 μM), nitrendipine (10 μM) and niflumic acid (100 μM).The NCX current was obtained as the nickel‐sensitive current by subtracting the current in the presence of 10 mM NiClto) was studied using a protocol consisting of a 30‐ms pre‐pulse from a holding potential of −80 to −40 mV to inactivate sodium channels followed by a 300‐ms test pulse to +60 mV in 10‐mV increments at a frequency of 0.1 Hz in the presence of 200 μM CdCl2 in Ca2+‐free normal Tyrode's solution as described previously to was measured as the difference between the peak outward current and the steady‐state current Ksus) was evaluated as the difference between the holding current and the end of the steady‐state current.The transient outward potassium current (IKur) was measured as 4‐aminopyridine‐sensitive currents. To record IKur without contamination by Ito, IKur was dissected by 1 mM 4‐aminopyridine with a double‐pulse protocol, consisting of a 100‐ms depolarizing pre‐pulse to +40 mV from a holding potential of −50 mV to inactivate Ito, followed by 150‐ms voltage steps from −40 to +60 mV in 10‐mV increments at room temperature to provide adequate temporal resolution The ultra‐rapid delayed rectifier potassium current  was monitored with a step‐pulse protocol . Once the IK became stable , apamin (100 nM) was applied, and a step‐pulse protocol was performed when the IK reached a steady state. The IKAS was calculated as the difference between the absence and presence of apamin. All experiments were performed at 36°C. The external solution contained (in mM): N‐methylglucamine, 140; KCl, 4; MgCl2, 1; glucose, 5; and HEPES, 10 (pH 7.4 with HCl) The I2+ ATPase , NCX , Cav1.2 , phospholamban , Thr17‐phosphorylated PLB , PLB‐Ser16 (Badrilla), Ca2+/calmodulin‐dependent protein kinase II , Kv1.4 (Alomone Labs), Kv1.5 (Alomone Labs), Kv4.2 (Abcam) and α‐actin . Blots were reacted with primary antibodies followed by horseradish peroxidase‐conjugated secondary antibodies. Bound antibodies were detected with an enhanced chemiluminescence (ECL) detection system of an ECL Plus Kit  and analysed with AlphaEaseFC software . Targeted bands were normalized to α‐actin.Control and colchicine‐treated HL‐1 cells were centrifuged and lysed using radioimmunoprecipitation (RIPA) buffer. The protein concentration was determined with a Bio‐Rad protein assay reagent . For the analysis, equal amounts of protein from each sample were separated using 4~16% Tris‐acetate polyacrylamide gradient gel electrophoresis. After electrophoresis, protein samples were transferred onto equilibrated polyvinylidene difluoride membranes . Blots were probed with primary antibodies against ryanodine receptor type 2 , RyR phosphorylation at S2808 and S2814 , SR Ca™ 4‐well chamber slides  for 24 hrs, were fixed in PBS containing 4% paraformaldehyde for 30 min., blocked with 3% bovine serum albumin and incubated in primary antibody used with anti α‐tubulin antibody  at a dilution of 1:200 for 2 hrs, and washed three times with PBS for 5 min. Cells were then incubated in secondary antibody used with FITC‐conjugated antibody  at a dilution of 1:200 for 1 hr. Finally, counterstaining of nuclei was performed with 4′, 6‐diamidino‐2‐phenylindole . The cells were observed and photographed with a Leica TCS SP5 Confocal Spectral Microscope Imaging System .HL‐1 cells with and without colchicine (3 nM) or collagen (10 μg/ml), cultured in Lab‐Tekt‐test was used to compare differences between control and colchicine‐treated HL‐1 cells and collagen‐treated HL‐1 cells with or without colchicine. Nominal variables were compared by a Pearson's chi‐square test or Fisher's exact test. A P value of <0.05 was considered statistically significant.All quantitative data are expressed as the mean ± standard error of the mean (S.E.M.). A paired 2+]i transients than control HL‐1 cells. Moreover, colchicine (3 nM)‐treated HL‐1 cells had 47% smaller caffeine‐induced [Ca2+]i transients than the control group, which suggests less SR Ca2+ stores in colchicine (3 nM)‐treated HL‐1 cells. Moreover, colchicine (3 nM)‐treated HL‐1 cells had smaller ICa‐L and outward (reverse mode) NCX current densities than control HL‐1 cells ‐treated HL‐1 cells. The colchicine (3 nM)‐treated HL‐1 cells also had lower expressions of total PLB and PLB‐Thr17 by 18% and 23%, but had similar expressions of PLB‐Ser16 ‐treated HL‐1 cells was lower by 14% and 11% than in the controls. Moreover, the expression of CaMKII, a multifunctional serine/threonine protein kinase that mediates Ca90, APD50 and APD20, but had similar values for the APA and RMP. In contrast to the effects of colchicine, collagen (10 μg/ml) shortened the APD90 in HL‐1 cells and had more delayed afterdepolarizations . Furthermore, colchicine (3 nM) ameliorated APD changes in collagen (10 mg/ml)‐treated HL‐1 cells as shown in Figure As shown in Figure 2+ homeostasis is essential for maintaining normal cardiac function and electrical activity. There is tight coupling between Ca2+ influx and Ca2+ release. Dysregulated Ca2+ handling can generate the triggered activity, commonly associated with AF development 2+ overload may potentially produce arrhythmogenesis in atrial myocytes 2+]i transients and SR Ca2+ content, which suggests colchicine decreases Ca2+ concentration in both cytoplasm and the SR. Accordingly, colchicine might directly modulate Ca2+ homeostasis in atrial myocytes and result in a decrease in the occurrence of AF. In contrast, previous studies on rat or chick embryos ventricular myocytes showed that microtubule disruption by colchicine  increased the ICa‐L and [Ca2+]i transients, but did not modify SR Ca2+ content to in isolated mouse or dog ventricular myocytes CaCa‐L and reverse mode NCX in HL‐1 cells, colchicine can down‐regulate SERCA2a, total and PLB‐Thr17. A decrease in PLB phosphorylation at Thr17 can functionally reduce SERCA2a Ca2+ reuptake by increasing the effects of PLB on the inhibition of SERCA2a activity. All these changes can result in a decrease in SR Ca2+ content and Ca2+ transients. Colchicine decreased CaMKII expression, which is a key regulator of excitation‐contraction coupling in cardiomyocytes. CaMKII activation may enhance ICa‐L facilitation and increase Ca2+ influx, which accompanies Ca2+ overload. In addition, CaMKII can enhance Ca2+ leakage to generate triggered activity. The potential of CaMKII inhibition to reduce an arrhythmogenic outcome was demonstrated in AF 2+ sparks from a previous study 2+ regulatory proteins, which may inhibit the occurrence of AF.In addition to reductions in Ito, and IKsus, which can overcome the APD‐shortening effect by ICa‐L reduction. Thus, colchicine modulates atrial electrophysiological property by interfering Ca2+ and potassium currents, which may ameliorate AF occurrence. Similarly, cytoskeletal disruption was also reported to increase the APD in ventricular myocytes by decreasing the Itoto and IKsus mediated by colchicine.We found that colchicine can increase the APD, which may reduce the generation of re‐entrant circuits and AF maintenance. This prolongation of the APD may be caused by a reduction of reverse mode NCX, Ito and IKsus2+ overload, which facilitates the occurrence of trigger activities in atrial and pulmonary vein myocytes. Colchicine normalized the APD in collagen‐treated HL‐1 cells, and this suggests that the ECM controls cytoskeletal mechanics in the development of cardiac remodelling, and colchicine may modulate the remodelling process and atrial arrhythmogenesis.The ECM of collagen causes atrial myocytes to exhibit a shorter APD by increasing the ICa‐L2+ dysregulation may increase arrhythmogenesis in pulmonary vein cardiomyocytes. Colchicine may reduce AF recurrence after pulmonary vein ablation There are several limitations of our study that must be acknowledged. This study demonstrated the effect of colchicine on microtubule disruption in HL‐1 cells, an atrial cell line from mice. However, the ultrastructure of HL‐1 cells, including the cytoskeleton, and microtubule stability are different from adult naïve cardiomyocytes. 2+ regulation, which may be responsible for preventing AF occurrence or recurrence. These changes also demonstrate that microtubule modulation may play an important role in the regulation of ionic channels and Ca2+ homeostasis, which contributes to the pathogenesis of AF.In conclusion, our findings suggest that colchicine modulates atrial electrical activity and CaThe authors confirm that there are no conflicts of interest.


Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants KD of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ KD of 180 nM was determined. Thus, quantitative [Ca2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca Regulation of the intracellular Ca2+ concentration ([Ca2+]i) is maintained by a variety of channels, exchangers and pumps located in the plasma membrane or in internal membranes. Thus, this Ca2+ signaling toolkit results in an appropriate stimulus-induced temporal and spatial intracellular Ca2+ pattern 2+]i and its dynamics is crucial for a better understanding of physiological processes as well as dysfunctions.The basis of all cellular processes and activities is the precise regulation of intracellular ion homeostasis. Calcium ions  and Asante Calcium Red (ACR) are newly developed Ca2+-sensitive fluorescent dyes . Due to their improved spectral properties with greatly enhanced brightness and a large Stokes shift allowing single-wavelength and ratiometric imaging, these dyes seem to be promising new Ca2+ indicators in vitro. Moreover, the suitability of ACR for 2P-FLIM recordings was successfully demonstrated in living tissue. Here, salivary glands of the American cockroach Periplaneta americana were used, representing a well-established model system for studying biogenic amine-regulated epithelial ion transport Another approach, circumventing the above-mentioned pitfalls, is fluorescence lifetime imaging microscopy (FLIM), which can also be combined with two-photon (2P) excitation in vitro measurements, the Calcium Calibration Buffer Kit #1   was used to prepare 11 solutions of varying Ca2+ concentrations [Ca2+]free . For investigating protein-dye interaction, Ca2+-free and Ca2+-saturated buffer solutions were prepared containing 18 mM NaCl, 2 mM MgCl2·6 H2O, 40 mM KCl, 80 mM K gluconate, 5 mM HEPES and 1% (m/v) bovine serum albumin (BSA) . Additionally, the Ca2+-free buffer solution contained 2 mM EGTA and the Ca2+-saturated buffer solution 2 mM Ca(NO3)2. The pH was adjusted to pH 7.3 with 1 N HCl.For +-salts of the Ca2+-sensitive dyes Asante Calcium Red  and Asante Calcium Green  were dissolved in 50 µL double-distilled water to obtain 0.5 mM and 0.4 mM stock solutions, respectively. These stocks were then diluted in the Ca2+ buffer solutions, reaching final concentrations of [ACR]  = 2.5 µM and [ACG]  = 0.9 µM, respectively.The K2·6 H2O, 2 mM CaCl2·2 H2O, 10 mM glucose and 10 mM Tris, pH 7.4. According to 2+-free stock solution of 0.5 M K2H2EGTA and a Ca2+-saturated stock solution of 1 M K2CaEGTA were prepared by potentiometric back titration for in situ calibration experiments. These stocks were then diluted in a buffer solution (160 mM NaCl and 10 mM Tris) to the final concentration of 10 mM K2H2EGTA and 10 mM K2CaEGTA calibration buffer solutions . By mixing K2H2EGTA and K2CaEGTA, various free Ca2+ concentrations [Ca2+]free could be obtained according to Cockroach physiological saline contained 160 mM NaCl, 10 mM KCl, 2 mM MgClI = 0.16) it could be determined through a Beers fitting procedure at The given EGTA dissociation constant in situ calibration experiments, the non-ionic surfactant Triton X-100  was used to equilibrate defined extracellular and intracellular Ca2+ concentrations 2+]free  and 0.1% Triton X-100 (v/v). A 10 mM stock solution of dopamine  in double-distilled water was prepared daily and dissolved in physiological saline immediately before an experiment to a final dopamine concentration of 1 µM. The acetoxymethyl (AM)-ester of ACR  was diluted in 27 µL Pluronic F-127 , divided into 1 µL aliquots and stored at −20°C. The aliquots were dissolved in hypotonic physiological saline  immediately before an experiment to the final dye concentration of 5 µM.For 2+-free and Ca2+-saturated buffer solutions were recorded. The dye concentration varied from 1.7 µM to 12.5 µM for ACR and from 0.9 µM to 1.8 µM for ACG. Fluorescence quantum yields of the Ca2+-saturated dye forms were determined absolutely with the C 9929 integration sphere system . Since the fluorescence quantum yields of the Ca2+-free dye forms were below the detection limit of this system (ΦF <0.01), fluorescence quantum yields of these dye forms were determined relative to the respective Ca2+-saturated form as a fluorescent reference ex = 550 nm with a repetition rate of 20 MHz and a pulse width of ∼30 ps. The laser beam was fiber-guided towards the fluorescence lifetime spectrometer FL920 , where the emitted fluorescence was detected by a multichannel plate  in the time-correlated single photon counting (TCSPC) mode.Absorption measurements were performed with a Lambda 750 UV/VIS spectrometer . To determine the absorption coefficients, absorption spectra in CaFσ2 were determined from relative measurements using the well-characterized 2P-reference rhodamine B in methanol 2+-free and Ca2+-saturated conditions, respectively. For 0.9 µM ACG, the rhodamine B concentration was adjusted to 10 nM and 5.0 µM in Ca2+-free and Ca2+-saturated conditions, respectively. The ΦFσ2 values with 10−50 cm4 s/photon  = 1 GM were calculated according to 2P fluorescence excitation action cross-sections Φc is the dye concentration, ΦF the fluorescence quantum yield, and r indicates the spectroscopic parameters of the 2P-reference rhodamine B. 2P-excitaiton was carried out by a Ti:Sa laser system  tuning the wavelength between λex,2P = 720 nm–900 nm, with a 82 MHz repetition rate and pulse width of ∼80 fs. The laser beam was coupled to the fluorescence lifetime spectrometer FL920  and was focused by a lens on the quartz cuvette. The fluorescence emission was detected with a photomultiplier  in a spectral range of 400 nm–700 nm in 1 nm steps. The obtained emission spectra were integrated, unraveling 2 values is estimated at 10%.P. americana (L.) was reared at 27°C under a light/dark cycle of 12 h: 12 h at the Department of Animal Physiology (University of Potsdam). The animals had free access to food and water. Only male adults aged between 4–6 weeks were used for experiments. Salivary glands were dissected in physiological saline as described previously A colony of the American cockroach Salivary gland lobes were incubated for 60 min in hypotonic saline containing 5.1 µM ACR/AM. After dye loading, the gland lobes were transferred to physiological saline for at least 10 min, then fixed on a coverslip coated with the tissue adhesive Vectabond Reagent  and mounted on a microscope stage. During the experiment, the gland lobes were continuously perfused with physiological saline at a flow rate of 2 ml/min.in vitro  and in situ imaging. The system includes an inverted microscope  equipped with an Olympus PlanApo ×100/NA1.4 oil-immersion objective. A mode-locked fs-fiber laser  was used as a 2P-excitation source, operating at λex,2P = 780 nm with a repetition rate of 50 MHz and a ∼90 fs pulse width. The laser beam was guided towards the objective via the microscope side port using a dichroic mirror . Fluorescence was guided through a 100 µm pinhole and was detected by a single-photon avalanche diode . For rejection of the excitation light, two short pass filters were used . Time-resolved fluorescence was performed in the TCSPC-mode by using a PicoHarp300  with a time resolution of 8 ps. Laser power was adjusted to achieve average photon counting rates of <105 photon/s and peak rates close to 106 photons/s, thus below the maximum counting rate allowed by the TCSPC electronics to avoid pulse pile-up. The image acquisition occurred by raster scanning the objective using a xy-piezo-positioner . Thereby, full-frame images of 80 µm×80 µm (150 pixel ×150 pixel) were acquired in around 30 s with a pixel dwell time of 0.6 ms. Data acquisition and analysis were performed by the SymPhoTime software version 5.3.2.2 . All photons collected in a distinct region of interest (ROI) were used to form a global histogram. The obtained decay curves were analyzed by a deconvolution fitting routine, whereby the quality of the fit was estimated by reduced MicroTime200  was used for time-resolved fluorescence measurements of P<0.05. Statistical analyses were performed using GraphPad Prism 4.01 . Graphical presentation was carried out using Origin 8 . Data are presented as the mean ± the standard error of the mean (SEM). The dissociation constants KD were calculated by fitting the data points to Statistically analyzed sets of data were distributed normally . Thus, the data sets were further analyzed by repeated-measures ANOVA and Holm-Sidak's multiple comparison post-hoc tests. Differences were considered statistically significant if v corresponds to fluorescence intensity and fluorescence decay time, respectively min refers to Ca2+-free buffer conditions and the subscript max to Ca2+-saturated buffer conditions.2+ chelating BAPTA ligand and seminaphtofluorescein as the fluorescent backbone, as published recently  to 542 nm (Ca2+-saturated)  to (47.8±0.8) 103 M −1 cm −1 . These absorption coefficients fit well with the published data 2+]free (2+-bound ACR at λex = 515 nm was determined absolutely at ΦF = 0.06±0.01 (N = 6), whereas it was estimated for the Ca2+-free ACR relatively at ΦF = 0.0017 as its fluorescence quantum yield was below the limit of detection to be determined by absolute measurements. Here, ACR in a Ca2+-saturated buffer was used as the fluorescent reference ex = 488 nm), a further fluorescence emission band around 525 nm appeared  . Concurr 2+]free . The fluappeared . However2+ chelating BAPTA ligand and a fluorescein derivative as the fluorescent backbone (2+-free) to 519 nm (Ca2+-saturated)  to (144±1.6) 103 M −1 cm −1 . Increasing [Ca2+]free caused a rise in the fluorescence intensity with an emission maximum at 542 nm. The fluorescence quantum yield of Ca2+-bound ACG at λex = 515 nm was determined absolutely at ΦF = 0.42±0.01 (N = 6), whereas it was estimated for the Ca2+-free ACG-relatively at ΦF = 0.0020 using ACG in a Ca2+-saturated buffer as the fluorescent reference. Presumably, for ACG as well as ACR, a photoinduced electron transfer is supposed to be the mechanism for changing fluorescence characteristics. Thereby, the Ca2+ complexation by BAPTA moiety blocks the electron transfer from the electron donor BAPTA to the fluorophore, which acts as an electron acceptor. Here, the fluorescence intensity also increases 2+-saturated and Ca2+-free buffers. Thereby, both dyes displayed a dependency of the FEF and the chosen excitation wavelength (ex = 548 nm and for ACG (231-fold) at λex = 525 nm. The excitation-dependent fluorescence enhancement for ACR has been described recently Analog to ACR, ACG is composed of a Cabackbone  [27]. Thturated) , whereasvelength . A maximKD,i were calculated using KD,i of 265 nM±33 nM (N = 4) is nearly twice as high as that of the manufacturer (135 nM) KD,i. Depending on the excitation wavelength, ACR can be used in either single wavelength  or dual wavelength  mode 2+]free. In this case, the ratio of the fluorescence intensities at 655 nm and 525 nm were used to calculate the corresponding KD,i = 415 nM±43 nM (N = 4). In the single wavelength mode of ACR, a KD,i = 463 nM±59 nM (N = 4) was calculated. In both cases, experimental data agree well with the manufacturer's data  KD,i values, ACG and ACR are both high-affinity Ca2+ indicators From steady-state measurements in commercial Calcium Calibration Buffer Kit #1, the dissociation constants F and 2P-absorption cross-section σ2. The latter was determined by relative measurements, whereby rhodamine B in methanol as well as the characterized 2P-reference was used F = 0.50±0.003; N = 5) was measured absolutely and is in good agreement with the published data ex,2P = 720 nm–860 nm the obtained 2P-absorption cross-sections σ2 of ACR were rather constant, and only increased slightly at longer wavelengths σ2  and 1.38 GM (Ca2+-bound) were determined . The third decay time component for both dyes was calculated at approximately 20 ps–30 ps. These short decay times were considered uncertain, since such short decay times cannot be reliably resolved with the present setup displaying an IRF of 220 ps (FWHM). Thus, ACR exhibited two distinct fluorescence decay time components most probably attributed to the Ca2+-free (τfree = 0.12 ns±0.006 ns) and the Ca2+-bound species (τbound = 0.57 ns±0.003 ns), respectively. For ACG, the decay time component of the Ca2+-free form is in the same range as that for ACR (τfree = 0.25 ns±0.04 ns). On the other hand, the decay time component of the Ca2+-bound form is considerably higher (τbound = 2.38 ns±0.02 ns). The stated errors of the calculated fluorescence decay times were fitting errors derived from the FAST fitting routine.The obtained fluorescence decay curves of the Caobserved . This coi showed an opposite trend due to changes in [Ca2+]free. Thereby, the normalized amplitudes αf of the short decay time component, corresponding to the Ca2+-free dye form, decreased when [Ca2+]free increased, whereas the normalized amplitudes αb of the long decay time component, corresponding to the Ca2+-bound dye form, rose due to increasing [Ca2+]free  to approximately 60 nM±5 nM and 9 nM±3 nM, respectively. The calculated KD,t values for both dyes are slightly lower than the measured one, but they are within the same order of magnitude. In general, the dissociation constant KD indicates the measurable analyte concentration range from approximately 0.1 to 10 times the KD. However, the apparent value of KD is determined by the particular measured parameter as it has been shown, for instance, for other Ca2+ indicator dyes recording fluorescence intensities or fluorescence decay times Thus, it is possible to estimate m/v)) in the in vitro buffer solutions resulted in a bathochromic shift in the Ca2+-free ACR absorption maximum from 537 nm to 555 nm, and increasing [Ca2+]free led to a further bathochromic shift from 555 nm to 563 nm  for the Ca2+-free (λex = 543 nm) and ΦF = 0.08 (N = 3) for the Ca2+-bound (λex = 550 nm) ACR species in 1% BSA in vitro buffer solutions. Obviously, the influence of BSA seemed to be stronger on the Ca2+-free than on the Ca2+-bound ACR species. In fact, the bathochromic shift in the absorption spectrum and the hypsochromic shift in the emission spectrum as well as enhanced intensity have also been reported for other Ca2+-sensitive dyes such as Fura-2, Indo-1 and Quin-2 2+-chelating side interact with proteins. Due to their higher affinity to Ca2+ ions the carboxyl group would be rather occupied by Ca2+ than by proteins. Thus, in the presence of Ca2+ the formation of a protein-dye complex is interfered.The fluorescence intensity of ACR under Cao 641 nm . In the  BSA vs.. This be2+-free and the Ca2+-bound species were about 5 times longer than those obtained in BSA-free environments and amounted to τfree = 0.77±0.002 ns (N = 3) and τbound = 2.86±0.04 ns (N = 3), respectively.The obtained fluorescence decay curves could be adequately fitted by a deconvolution biexponential fitting routine yielding reasonable residuals and  values . The detex,2P = 780 nm as described previously, and yielded σ2 = 40 GM±1 GM (N = 3) for the Ca2+-free and σ2 = 24 GM ±1 GM (N = 3) for the Ca2+-bound dye species. Nevertheless, the 2P-action cross-section ΦFσ2 of the Ca2+-free and Ca2+-bound dye species were calculated to 0.4 GM and 1.9 GM, still indicating an increase in the ACR fluorescence intensity due to increasing [Ca2+]free in the presence of BSA. All spectroscopic parameters determined in BSA-containing buffer solutions deviated from those perceived in BSA-free calibration buffer solutions, which can be attributed to dye-protein interactions. This fact underlines the need for determining the calibration parameters used for intracellular ion concentration quantification individually in each biological system.The 2P-absorption cross-sections of ACR in the presence of BSA were determined at λin vitro measurements of ACG have unraveled larger absorption coefficients, higher fluorescence quantum yields, and larger FEFs compared to those of ACR, at this time ACG is still only commercially available in K+-salt form, but not in the ester form required for experiments in living tissues. Indeed, the ACG salt form could be microinjected into a selected living cell for in situ recordings. However, this would be an invasive approach and the simultaneous analysis of several cells would be virtually impossible. On the other hand, ACR is commercially available as an AM-ester. So, subsequently the feasibility of ACR for 2P-FLIM recordings in living cells was tested. The investigated salivary gland ducts showed a relatively low cellular autofluorescence after 2P-excitation at λex,2P  = 780 nm, but with a distinct signal induced by the luminal cuticle  and for 30 min at an increased acquisition rate of 1.3 min−1 (P = 3.7 mW). In the first case, the initial fluorescence intensity is reduced by one-third after 60 min, which can presumably be attributed to dye leakage  and Ca2+-saturated  conditions, respectively in situ measurements was tested in duct cells of cockroach salivary glands, representing a well-established model system for investigating aminergic controlled transepithelial ion transport processes 2+]i in the duct cells from 48 nM±4 nM to 311 nM±43 nM as demonstrated by steady-state fluorescence recordings using Fura-2 2+]i elevation 2+]i increase from 144 nM±15 nM to 372 nM±160 nM was confirmed using the Ca2+ sensor dye OGB-1 for 2P-FLIM recordings 2+ signaling response is a valuable tool for testing new Ca2+ sensor dyes in situ. When [Ca2+]i increases, the corresponding ACR τav,amp should increase as well. To verify this assumption, ACR-loaded salivary duct cells were stimulated twice with 1 µM dopamine, and 2P-FLIM images were recorded continually . The appropriate fluorescence decay curves were fitted by a biexpontential decay function with fixed decay time components from the in situ calibration. Based on the fitting results, the normalized amplitudes αi as well as τav,amp values were calculated . In the presence of dopamine, [Ca2+]i increased to 590 nM±300 nM (N = 15). The second dopamine stimulus resulted in a similar rise to 500 nM±260 nM (N = 15)  . The des5)  was recorded in aqueous buffer solutions of varying [Ca2+]free from 0 µM–40 µM. The inset shows the [Ca2+]free dependent normalized fluorescence intensity at the emission wavelengths λem = 525 nm (blue triangles) and λem = 650 nm (red triangles).(TIF)Click here for additional data file.Figure S2Leakage and photobleaching of ACR in salivary duct cells. Salivary gland lobes were incubated with 5.1 µM ACR/AM for 60 min. After acclimatization, 2P-FLIM images were recorded at λ2P-ex = 780 nm. (A) Low image acquisition rate  presumably indicates dye leakage from the cells . Fit to monoexponential decay function yielded a leakage half-time of t1/2 = 61 min (dashed black curve). High image acquisition rate  presumably indicates dye photobleaching . Fit to monoexponential decay function yielded a photobleaching half-time of t1/2 = 14 min (solid black curve).(TIF)Click here for additional data file.


Because the Ca2+-buffering protein calreticulin (CRT) is able to bind and sequester Ca2+, it can serve as a mobile intracellular store of easily releasable Ca2+ and control its local concentration within the cytoplasm. Our previous studies showed an enhanced expression of Petunia hybrida CRT gene (PhCRT) during pistil transmitting tract maturation, pollen germination and tube outgrowth on the stigma, gamete fusion, and early embryogenesis. Here, we demonstrate that elevated expression of CRT results in the accumulation of this protein in response to anthesis, pollination, sperm cells deposition within the receptive synergid and fertilization, when the level of exchangeable Ca2+ changes dynamically. CRT localizes mainly to the endoplasmic reticulum and Golgi compartments in the pistil transmitting tract cells, germinated pollen/tubes, and sporophytic/gametophytic cells of the ovule and corresponds with loosely bound Ca2+. Additionally, the immunogold research shows, for the first time, highly selective CRT distribution in specific nuclear sub-domains. On the basis of our results, we discuss the possible functions of CRT with respect to the critical role of Ca2+ homeostasis during key events of the multi-step process of generative reproduction in angiosperms.Calcium (Ca Stored Ca2+ is sequestered in different cell compartments  and specific organelles (such as the endoplasmic reticulum(ER)) where it can be associated with specific proteins that buffer and release Ca2+ to control its local concentration. This pool of Ca2+ is exchangeable and can transform into the other forms when and where it is needed. Different Ca2+ forms function in interactions of the male gametophyte  with the female sporophytic tissues (pistil transmitting tract and ovular somatic tissues) and female gametophyte (the embryo sac)  for 4 h at room temperature, and then subsequently postfixed with 1 % (v/v) osmium tetroxide (OsO4) in the same buffer-antimonate solution for 12 h at 4 °C. Next, samples were dehydrated in graduated ethanol concentrations and embedded in Poly/Bed 812 resin (Polysciences) according to the standard protocol. Ultra-thin longitudinal sections were collected on copper grids, stained with 2.5 % (w/v) uranyl acetate and 0.4 % (w/v) lead citrate solutions, and examined by transmission electron microscopy (Jeol EM 1010) at 80 kV. The presence of Ca2+ in the precipitates was confirmed previously using energy-dispersive X-ray microanalysis  potassium antimonate, 2 % (v/v) glutaraldehyde, and 2 % (v/v) formaldehyde in 0.1 M phosphate buffer , 1 mM EGTA, 2 mM DTT plus 1 mM PMSF and cOmplete Protease Inhibitor Cocktail (Roche) according to the manufacturer’s recommendation. The homogenates were centrifuged at 16,000g for 30 min at 4 °C. Protein concentrations of the supernatants were measured with the Bio-Rad DC Protein Assay according to the manufacturer’s instructions. Equal amounts of proteins were separated by electrophoresis on a 12.0 % SDS–PAGE gel and then the proteins were semi-dry transferred to Amersham PVDF Hybond-P membrane . Blocked blots were probed with a rabbit polyclonal antibody against maize CRT (CRT PAb)  and then with anti-goat IgG-HRP secondary antibody (Santa Cruz Biotechnology). Detection was performed as described above. Each Western blot was performed a minimum of three times for each experiment, and representative blots shown. Quantification of signals was done with Image Gauge 3.4 software (Science Lab99). Statistical significance of data was determined by a one-way ANOVA test.100 mg of whole The samples of stigmas and ovules dissected from pollinated pistils were fixed with 4 % (v/v) formaldehyde and 0.25 % (v/v) glutaraldehyde in phosphate-buffered saline  for 1 h at room temperature (slight vacuum infiltration) followed by overnight fixation at 4 °C. Fixed samples were dehydrated in graduated ethanol concentrations, embedded in LR Gold resin (Fluka) according to the standard protocol, and ultra-thin longitudinal sections were collected on Formvar-coated nickel grids. The sections were then incubated with blocking solution containing 3 % (w/v) bovine serum albumin (BSA) in PBS buffer, pH 7.2, for 5 min at room temperature, incubated in 1:20 dilution of a primary CRT PAb in PBS supplemented with 0.3 % (w/v) BSA for 2 h at room temperature, and then incubated with gold-conjugated goat anti-rabbit IgG antibody , diluted 1:100 in PBS buffer with 0.2 % (w/v) BSA for 1.5 h at room temperature. In the control, incubation with the CRT PAb was omitted. Finally, the sections were stained with 2.5 % (w/v) uranyl acetate and 0.4 % (w/v) lead citrate solutions and examined by transmission electron microscopy as above.Petunia pistil  in the cytoplasm of stigma transmitting tract (sitt) cells  of sitt  anthesis and pollination resulting in pollen germination and tube outgrowth on the stigma, and (2) pollen tube entry into the receptive synergid, resulting in gamete fusion and embryogenesis.Here we have demonstrated that both CRT and exchangeable Ca2+ and CRT during the complicated intercellular communication that occurs between the male gametophyte and the female sporophyte/gametophyte cells. Although both external and internal Ca2+ stores are likely important in this process, most previous research has focused on the essential role of the ecm in Ca2+ storage and mobilization during pollen–pistil interactions. The ER and Golgi stacks are known to be the effective intracellular Ca2+ stores -mediated Ca2+ signaling were found in both the nuclear envelope and the NR of animal cells. Existence of such Ca2+-signaling machinery has also been confirmed in purified tobacco nuclei. InsP3 induces release of Ca2+ from both the nuclear envelope and the NR into the nucleoplasm, resulting in generation of physiologically important Ca2+ signals that can be recognized by Ca2+-binding proteins involved in signaling. This is also a mechanism of maintaining nuclear Ca2+ homeostasis  signals and is usually found primarily in the ER. However, the rough ER is continuous with the outer nuclear membrane, and some authors suggest a possible function of CRT in the lumen of the ER/nuclear envelope rather than in the nucleoplasm. By contrast, our present immunogold research shows, for the first time, highly selective CRT localization in specific nuclear sub-domains. How CRT could leave the ER and move into the nucleus remains unknown. Possible explanations include production of splice variants that can localize to both places, posttranslational modifications such as glycosylation, degradation of the C-domain containing HDEL, or enzymatic modification of the glycan complexity (see reviews by Johnson et al. 2+ homeostasis during anthesis and pollen–pistil interactions in plants depends on intracellular Ca2+ stores and CRT. CRT’s Ca2+-binding/buffering property is probably essential to control mobilization of stored Ca2+ from the ER, Golgi compartments, and nuclei. We consider the possibility that CRT enters nuclei only during certain events, such as generative reproduction in plants. Additionally, CRT’s role as a molecular chaperone may be equally important because a high rate of protein synthesis is required during the multi-step process of plant sexual reproduction. Future functional work will help us sort out these possibilities.In conclusion, our results suggest that the precise regulation of Ca