Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+]i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from −80 to −40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca Conductchannels , Ca2+ entraction . The occ by Ano1 . While ie in ICC , the mec regions . The plas in ICC  that caus in ICC , but the+/Ca2+ exchanger is typically considered a Ca2+ extrusion transporter, but the exchanger is capable of bi-directional movement of ions depending upon the membrane potential and the transmembrane gradients for Ca2+ and Na+  in ICC isolated from the murine colon and small intestine and purified by FACS. We compared expression of Slc8a1-3 transcripts in ICC and in the mixed cell population obtained from enzymatic dispersion of muscles. We found that NCX3 is associated with ANO1 channels, and we evaluated the function of NCX in activating and sustaining activation of CaCC during slow wave currents using the patch clamp techniques on single ICC and imaging of Ca2+ transients in intact networks of pacemaker ICC.In the present study we evaluated expression of KitcopGFP/+ mice, described previously . For imaging experiments Ai95 (RCL-GCaMP6f)-D (GCaMP6f mice) and their wild-type siblings (C57BL/6) were purchased from the Jackson Laboratory . B6. c-Kit+/Cre–ERT2 (Kit-Cre mice) were gifted from Dr. Dieter Saur of the Technical University Munich, Germany. GCaMP6f mice were crossed with Kit-Cre mice, and the resulting offspring are referred to as Kit-Cre-GCaMP6f mice throughout the manuscript. These mice were injected with tamoxifen at 6–8 weeks of age to induce Cre Recombinase and activate expression of GCaMP6f. Tamoxifen  was dissolved in 800 μL of ethanol  by vortexing for 20 min. Then 3.2 ml of Safflower (generic) was added to create solutions of 20 mg/ml, which were then sonicated for 30 min prior to injection. Mice were injected  with 0.1 ml of tamoxifen solution (2 mg tamoxifen) for three consecutive days. Mice were used for experiments 10 days after the first injection. Expression of GCaMP6f was confirmed by genotyping and imaging.C57BL/6  and eviously , were usOn the days of experiments animals were anesthetized with isoflurane  prior to decapitation and then small intestines and colon were removed. All animals used and the protocols performed throughout this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The institutional Animal Use and Care Committee at the University of Nevada approved all procedures used in the breeding and killing of animals.copGFP/+ mice, as described previously (2+-free Hanks’ solution for 20 min. The muscles were incubated in a Ca2+-free enzyme solution containing (per ml): collagenase , bovine serum albumin , trypsin inhibitor  and ATP (0.27 mg). Cells were plated onto coverslips coated with murine collagen . The cells were allowed to stabilize for 2 to 6 h at 37°C in an incubator (atmosphere 95% O2–5% CO2) in culture media  supplemented with 2% antibiotic–antimycotic  and stem cell factor .Interstitial cells of Cajal were isolated from Kiteviously . Briefly2+-free enzyme solution containing (per ml): collagenase , bovine serum albumin (8 mg), trypsin inhibitor (8 mg), papain (1 mg), and L-dithiothreitol . After incubation in the enzyme solution for 30 min, SMCs liberated by trituration and stored in Ca2+-free Hanks’ solution at 4°C for no more than 6 hrs.To isolate SMCs from proximal colon , the orgKitcopGFP/+ mice were purified by fluorescence-activated cell sorting  using 488 nm excitation and a 530/30-nm bandpass filter for GFP, as previously described (Hprt) standard after log transformation of the corresponding raw data.Interstitial cells of Cajal from escribed . Total RInterstitial cells of Cajal were identified as cells with green fluorescent protein using an inverted fluorescence microscope. The standard whole-cell patch clamp configuration was employed to record membrane currents (voltage clamp mode). Currents were amplified with an Axopatch 200B patch-clamp amplifier  and digitized with a 16-bit analog to digital converter  and stored using pCLAMP software . Data were sampled at 4 kHz and filtered at 2 kHz using an eight-pole Bessel filter for whole-cell experiments. All data were analyzed using Clampfit  and Graphpad Prism  software.2+ currents were recorded from SMCs using amphotericin perforated whole cell patch clamp configuration. The stock solution of 90 mg/ml Amphotericin B (Sigma) was dissolved with dimethyl sulfoxide (DMSO) using sonication and diluted to a final concentration of 250 μg/ml in the pipette solution. STIC amplitude was measured using threshold events detection analysis in Clampfit. Ten seconds of each recording were analyzed. The minimum threshold amplitude of STICs was set to 6 pA and the averaged amplitude of all events from one recording was displayed as n = 1. The number of events in 10 s was used to determine the frequency of STICs . The amplitudes of currents in each cell activated by KB-R7943 or SN-6 at 0 mV were normalized to its cell capacitance . The tail currents of slow wave currents were obtained by repolarization from −40 to −80 mV. The durations of tail currents were measured from the peak amplitude of inward tail currents to the end of tail currents .L-type Ca2+-containing physiological salt solution  and (2) ECl = −40 mV solution (Solution VI). In some experiments, NaCl (0 and 20 mM) of external solution were replaced with equimolar LiCl (Solution II and III). For measurement of ANO1 currents expressed in HEK 293 cell, an external solution was Solution IV and internal solution was Solution VII. For Cav3.2 and L-type Ca2+ currents, external and internal solutions were Solution I and Solution V, respectively.The external solution for whole-cell recordings was a Ca2+ imaging was performed in situ with an Eclipse E600FN microscope  equipped with a 60 × 1.0 CFI Fluor lens . GCaMP6f was excited at 488 nm . The pixel size using this acquisition configuration was 0.225 μm. Image sequences were collected at 33 fps with TILLvisION software . Imaging was performed in the presence of nicardipine (100 nM) to reduce contractile movements and any residual movements were stabilized digitally with Volumetry software prior to analysis of Ca2+ transients. For experiments involving pharmacological treatments, control video sequences were collected for 20–30 s, and then KRB solution containing the drug concentration to be tested was perfused into the bath for 12–15 min before another 20–30 s period of imaging was performed.Flat sheets of jejunum muscles were pinned to the bottom of a 60 mm dish coated with Sylgard elastomer , with the longitudinal muscle side facing upward. The dish was continuously perfused with warmed KRB solution (37°C) for 1 h before experimentation. Following this equilibration period, Ca2+ transients in myenteric ICC (ICC-MY) were quantified using particle (PTCL) analysis, as described previously (t = ± 66–70 ms) and Gaussian filter  was applied to distinguish Ca2+ transients from the background. A particle analysis routine was applied by using of a flood-fill algorithm that marked the structure of all adjoining pixels that had intensities above the threshold. Ca2+ transient PTCLs were brighter and larger than noise particles. The threshold at which noise particles emerged and reduced the average particles size was thresholded and then valid Ca2+ PTCLs, above this threshold, were saved as a coordinate based PTCL movie. Any remaining noise in the PTCL file was removed by only including PTCLs > 6 μm2  in analysis. Ca2+ transients in ICC-MY occurred in temporal clusters  of multiple firing sites in the ICC-MY network firing asynchronously within a ∼1 s window as previously described , centrifuged at 16,000 × g at 4°C for 10 min. The 16,000 × g supernatant was centrifuged at 100,000 × g for 1 h to obtain the membrane fraction . The pellet was resuspended into IP/lysis buffer , and protein concentrations were determined by a Bradford assay using bovine g-globulin as the standard. ANO1, NCX3, and rabbit IgG antibodies (AB-105C)  (5 μg) were crosslinked to Protein-A/G-magnetic beads using the Pierce Crosslink Magnetic IP/Co-IP kit according to the manufacturer’s instructions . Membrane fractions (500 μg) were incubated with either rabbit anti-NCX3 specific antibody (AP12808b) , or rabbit anti-ANO1 antibody (ab64085)  overnight with rotation at 4°C. Captured proteins were eluted with low pH buffer. Eluted proteins were analyzed using the anti-NCX3 or the anti-ANO1 antibodies with ProteinSimple Wes (Co-immunoprecipitation (Co-IP) studies were performed to investigate possible protein-protein interactions between NCX3 and ANO1. Small intestine smooth muscle tissues were snap-frozen in liquid nitrogen and stored at −80°C after removal of the mucosa and sub-mucosa. Muscles were homogenized in lysis buffer  for 4 h, fixed in 4% paraformaldehyde for 4 min, and then permeabilized and blocked with PBS containing 0.2% tween-20 and 1% bovine serum albumin for 10 min at room temperature. PLA was performed following the manufacturer’s instructions . The fixed and permeabilized cells were incubated with goat anti-NCX3 antibody (1:200 dilution)  for 1 h, at room temperature, washed three times with PBS and then incubated with the rabbit anti-ANO1 antibody (1:200 dilution) for 1 h at room temperature. The slides were washed three times with PBS and then incubated with the Duolink minus-anti rabbit IgG and plus-anti goat IgG secondary antibodies (1:5 dilution) at 37°C for 1 h, followed by the ligation and amplification reactions . Finally, mounting medium with DAPI was used. The slides were examined using a LSM510 Meta  or Fluoview FV1000 confocal microscope . Confocal micrographs are digital composites of the Z-series of scans . Settings were fixed at the beginning of both acquisition and analysis steps and were unchanged. Final images were constructed using FV10-ASW 2.1 software (Olympus). As a negative control (two proteins not in close proximity), primary rabbit anti-ANO1 and mouse anti-myosin light chain (sc-48414)  antibodies were used, followed the by plus-anti rabbit IgG and minus-anti mouse IgG secondary antibodies, and the ligation and amplification reactions. The number of PLA spots per cell was divided by cell area, and these values were normalized to obtain PLA density values. Cell area was calculated with Image J software (NIH).Isolated ICC were incubated on slides coated with murine collagen (2.5 mg mlAno1 (A variant) was subcloned into pcDNA3.1 (Invitrogen). Ano1 was ligated into the pmKate2-N vector , containing the red fluorescent protein mKate2. The Ano1 stop codon was removed and EcoRI and Apa1 restriction sites were placed at the termini by PCR, so the Ano1 transcript was inserted in frame and resulted in a plasmid encoding a C-terminal mKate2 tagged ANO1 fusion protein. An Ano1 splice variant, AC, that is expressed in gastrointestinal muscles (Ano1 coding region using a QuickChange XL site-directed mutagenesis kit (Agilent Technologies). The plasmids were sequenced by the Nevada Genomics Centre to confirm insertion of the alternative exon. Ano1 (AC splice variant) was expressed in human embryonic kidney (HEK) 293 cells . HEK 293 cells were seeded in 12-well plates and maintained in DMEM (Gibco) medium with FBS , penicillin–streptomycin  and glutamax . The plasmid containing Ano1 tagged with mKate2 (0.5 μg/well) was transfected into cells using 1.5 μl/well of FuGENE 6 transfection reagent . Expression of Ano1 was confirmed by monitoring mKate2 fluorescence in cells.An expressed sequence tag (IMAGE Consortium cDNA clone number 30547439) homologous to murine  muscles , was genv3.2 channels were donated from Dr. E. Perez-Reyes . Generation of the cell lines and the electrophysiological properties of Cav3.2 currents were described previously (v3.2 (CACNA1H) was transfected into HEK 293 cells  with vector . Cells were grown in 15 mM HEPES D-MEM/F-12 media  supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% (v/v) pencillin-streptomycin (Gibco) at 37°C in 95% O2-5% CO2.HEK-293 cell lines expressing human Caeviously . Human C2+ imaging were perfused constantly with KRB solution containing (mmol/L): NaCl, 120.35; KCl, 5.9; NaHCO3, 15.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; and glucose, 11.5. KRB solution was bubbled with a mixture of 97% O2 – 3% CO2 and warmed to 37 ± 0.2°C. Drugs used to inhibit CaCC were 5-nitro-2-(3-phenylpropylamino)-benzoic acid acetamide . Drugs States); ] and 2-. NPPB . In all statistical analyses, p < 0.05 was considered statistically significant.Data are expressed as means ± SEM of n cells for patch clamp experiments and Slc8a1, Slc8a2, and Slc8a3 were analyzed in ICC and enzymatically dispersed, but unsorted, cells from the tunica muscularis of the small intestine and colon. Previously we showed that ICC collected by FACS, as performed in this study, showed minimal expression of Pdgfra (a marker for fibroblast-like cells), Myh11 (a marker for SMC) and Uchl1 (a marker for neurons) and elevated levels of Kit transcripts (ICC marker) relative to the unsorted cells, suggesting that sorted ICC were highly purified by FACS  was estimated to be −0.4 mV using the equation ENCX = 3ENa – 2ECa at 30°C . KB-R7943 increased currents at 0 mV from 2.9 ± 0.9 to 29.9 ± 2.8 pA . n = 5).Under the basal conditions of our experiments, the reversal potential for NCX  on STICs by reducing extracellular Na+ ([Na+]o). Cells were held at −80 mV. After STICs were recorded under control conditions , [Na+]o was replaced with 140 mM Li+ (Solution II), which would be predicted to shift ENCX to very negative potentials. Replacement of [Na+]o increased the frequency and amplitude of STICs from 58 ± 18 cpm to 111 ± 17 cpm . STICs reversed at −28 ± 2 mV (n = 5) when cells were dialyzed with Solution VI . These data suggest that STICs, activated by reducing [Na+]o, were due to a Cl– conductance and reversed at the calculated ECl.Interstitial cells of Cajal generate STICs that are thought to be the basal pacemaker activity in ICC . We test to 0 mV , n = 5. 2+ entry mode of NCX, Ca2+ entering cells may increase [Ca2+]i and activate CaCC. We have previously shown that Ano1 transcripts and protein are highly expressed in ICC (+]o are sustained for periods required to test CaCC inhibitors. Replacing [Na+]o (Solution II) increased STICs, as above, and the activation of STICs was sustained for at least 10 min  and SN-6 (5 μM) significantly reduced the amplitude of STICs in colonic ICC  on STICs after reduced o (Solution III) to −688 ± 150 pA (p < 0.05 vs. control). SN-6 (5 μM) decreased the peak amplitude of slow wave current to −51 ± 7 pA (p < 0.01 as compared to the amplitude with 20 mM [Na+]o)  . Tail cu [Na+]o) .n = 5 each).Another interpretation of the effects of SN-6 and KB-R7943 on STICs and slow wave currents could be a direct blocking effect of these drugs on the channels involved in these currents. Therefore, we performed control experiments to determine if KB-R7943 and SN-6 block ANO1 currents, which contribute to both STICs and slow wave currents. HEK 293 cells expressing ANO1 channels were stepped from −80 to +80 mV under whole cell voltage clamp conditions (Solution IV for bath solution and Solution VII for pipette solution). After obtaining control currents, the cells were exposed to KB-R7943 (5 μM) or SN-6 (5 μM) and the voltage clamp protocol was repeated. Neither of these agents had resolvable effects on ANO1 currents, as shown by the raw current traces  or the I2+ currents, since these conductances are present in ICC and may help organize the openings of ANO1 channels into slow wave currents (V3.2 currents were recorded in whole cell voltage-clamp experiments from HEK 293 cells transfected with CACNA1H. Cells were exposed to CaPSS (Solution I) and dialyzed with Cs+-rich solution (Solution V) and stepped repeatedly from −80 to −40 mV. KB-R7943 (5 μM) decreased the amplitude of the inward current evoked by depolarization from −496 ± 79 to −261 ± 44 pA  . Under control conditions, ICC-MY firing rhythmic CTCs at a frequency of 27.2 ± 2.6 min–1 (n = 6). SN-6 (10 μM) did not change the frequency of CTCs , but the heat maps of CTC activity indicated that the total CTC Ca2+ signal was decreased by SN-6 . The raw values of CTC duration were exampled and plotted as a frequency histogram .Pacemaker activity in the small intestine arises from Ca(ICC-MY) . Ca2+ sirs, CTCs . We imag.6 min–1 , n = 6.  by SN-6 . Occurre of SN-6 . Close e of SN-6 . The ave of SN-6 , n = 6. istogram . The his+ gradient, due to Na+ entry from the action of the Na+K+Cl– cotransporter (NKCC1), may facilitate slow wave currents due to the Ca2+ entry mode of NCX (+ gradient is likely to occur via the actions of Na+K+ ATPase (Na+ pump). A gene array study performed on ICC found an average 6.5 fold higher expression of Atp1a2 in ICC-MY in the small intestine vs. whole tissue , tail currents after stepping back to −80 mV , and durations of tail currents . Data from these experiments are summarized in Modeling has suggested that a shift in the Nae of NCX . Restorae tissue . Atp1a2 of 27 nM . TherefoCo-immunoprecipitation assays, followed by Wes analysis of the immunoprecipitates, were utilized to investigate possible interactions between ANO1 and NCX3 proteins. in situ was investigated by PLA. ICC were exposed to ANO1 and NCX3 antibodies, each from a different host species, followed by the appropriate secondary antibodies each linked to + and – complementary oligos to capture the fluorophore probe . In the negative control, the PLA procedure was carried out on ICC exposed to anti-ANO1 and a myosin regulatory light chain antibody; two proteins expressed in ICC but located in distinct subcellular compartments (p < 0.001).The proximity of ANO1 and NCX3 in small intestine ICC artments . As show2+ entry and Ca2+ release in the initiation of pacemaker activity in ICC  or results in sustained Ca2+ entry (as in the stomach) via activation of L-type Ca2+ channels in SMCs  during membrane potential recording (current clamp conditions) . STICs aeceptors ] and actrs . Reduced extracellular Na+, a maneuver to shift the reversal potential for NCX, enhanced STICs. KB-R7943 and SN-6 reduced the STICs enhanced by low extracellular Na+, but did not block these events. This observation suggests that KB-R7943 and SN-6 did not block Ca2+ release from stores. Voltage-dependent Ca2+ currents (via T-channels) coordinate Ca2+ release from stores in ICC and thus control the openings of ANO1 channels that are responsible for slow wave currents  was shown to inhibit slow wave currents in ICC (V3.2) currents , thus favoring the Ca2+ exit mode at the resting potentials of ICC. Shifting ENCX to negative potentials by reducing extracellular Na+ increased STICs at negative holding potentials. This observation suggests that NCX can flip to the Ca2+ entry mode and link to Ca2+-induced Ca2+ release to enhance STICs.Based on the ionic conditions of our experiments and assuming low initial Na2+ remaining elevated, during the plateau phase of slow waves has not been determined. Our data suggest that the longevity of the plateau phase is related to the duration of time that NCX remains in Ca2+ entry mode. Shifting ENCX of NCX to more negative potentials and creating a greater driving force for Ca2+ entry mode increased the duration of slow wave currents, and an inhibitor of NCX (SN-6) reduced the duration of slow wave currents and CTCs in ICC-MY in situ. When NCX switches to Ca2+ entry mode, the close association between NCX3 and ANO1 may be important for delivering Ca2+ to Ca2+ release channels in the ER to initiate Ca2+ transients and activation of ANO1 channels in the plasma membrane.STICs occur in a stochastic manner in ICC, but CaCC openings synchronize periodically to develop slow wave currents . Slow wa2+ entry mode during the course of the slow wave cycle is of interest. Activation of CaCC during slow wave currents causes Cl– efflux. ANO1 channels appear to be localized in microdomains in ICC  in this process   . Thus, cmodeling , the cha+ pump has been localized in microdomains formed by junctions between the plasma membrane and the sarcoplasmic reticulum in cardiac and SMC  is fundamental to the pacemaker activity of ICC.The α2 isoform of the Na and SMC . NCX exc and SMC , suggestENCX can be manipulated in single cells. In murine small intestine KB-R7943 reduced the amplitude and frequency of Ca2+ transients in networks of ICC in intact muscles. These authors suggested that NCX, working in Ca2+ entry mode, was responsible for Ca2+ store reloading. However, as shown by our control studies of the effects of KB-R7943 on voltage-dependent Ca2+ conductances, the reduction in slow waves by this compound could have been due to effects on voltage-dependent Ca2+ entry, an essential step in generation of slow waves , and no obvious role was identified for the Ca2+ exit mode of NCX in urethral interstitial cells. Inhibiting NCX under basal conditions inhibited STICs and Ca2+ transients suggesting these events are supported by Ca2+ influx due to the Ca2+ entry mode NCX. It should be noted that when membrane potential was clamped positive and negative to the estimated ENCX, there was no significant difference in STIC generation. This suggests that either the ENCX estimates were incorrect or that NCX, operating in Ca2+ entry mode is not essential for either generation of STICs  or maintenance of STICs by store refilling. Our observations suggest a dynamic nature for NCX in the slow wave cycle in which Ca2+ exit mode helps to maintain basal [Ca2+]i between slow waves and deactivate ANO1 at the end of the slow wave plateau, and Ca2+ entry mode sustains activation of ANO1 channels during the plateau phase of slow waves.A role for NCX in pacemaker activity has been suggested in studies of interstitial cells of the urethra  and smalow waves . In uretnd STICs , consist2+], [Na+], [Cl–], Cl– channels, Ca2+ release channels, NCX, NKCC1, Na+/K+ ATPase, and other transporters important for pacemaker activity and the development of slow wave currents may be focalized in microdomains, and relatively small ion fluxes may lead to significant concentration changes in these microdomains, as modeling has predicted  that flip NCX into Ca2+ entry mode. Ca2+ entry facilitates Ca2+-induced Ca2+ release, the sustained occurrence of Ca2+ transients , and sustains activation of ANO1, creating the plateau phase. In this study we have demonstrated a mechanism to explain how NCX might transition from Ca2+ exit to Ca2+ entry mode, and then how NCX might flip back to Ca2+ exit mode as a means of terminating slow wave currents and initiating slow wave repolarization. We have also observed a novel protein-protein interaction between NCX3 and ANO1 channels that might facilitate regulation of STICs and slow wave currents by NCX.In summary, our results suggest that the bidirectional nature of NCX is exploited during the slow wave cycle in ICC to maintain low levels of [CaThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Animal Use and Care Committee at the University of Nevada.2+ imaging experiments. BP and YX planned and performed the co-IP and proximity ligation assays. KO’D generated clones and vectors for expression of ANO1 channels in HEK 293 cells. SK and KS conceived of the hypothesis, collaborated in planning experiments, analyzed the data, and wrote and edited the manuscript.HZ planned, performed, and analyzed the electrophysiological experiments, collaborated on co-immunoprecipitation experiments, wrote the first draft of the manuscript, and edited the subsequent drafts. MZ performed and analyzed a portion of the electrophysiological experiments. BD and SB performed and analyzed CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Protein tyrosine kinases have been recognized as important actors of cell transformation and cancer progression, since their discovery as products of viral oncogenes. SRC-family kinases (SFKs) play crucial roles in normal hematopoiesis. Not surprisingly, they are hyperactivated and are essential for membrane receptor downstream signaling in hematological malignancies such as acute myeloid leukemia (AML) and mastocytosis. The precise roles of SFKs are difficult to delineate due to the number of substrates, the functional redundancy among members, and the use of tools that are not selective. Yet, a large num ber of studies have accumulated evidence to support that SFKs are rational therapeutic targets in AML and mastocytosis. These two pathologies are regulated by two related receptor tyrosine kinases, which are well known in the field of hematology: FLT3 and KIT. FLT3 is one of the most frequently mutated genes in AML, while KIT oncogenic mutations occur in 80–90% of mastocytosis. Studies on oncogenic FLT3 and KIT signaling have shed light on specific roles for members of the SFK family. This review highlights the central roles of SFKs in AML and mastocytosis, and their interconnection with FLT3 and KIT oncoproteins. The SRC-family of kinases (SFKs) comprises eight members: BLK, FGR, FYN, HCK, LCK, LYN, SRC and YES. They are further divided into two subfamilies: the SRC-related subfamily, also called Src-A , and the LYN-related subfamily, also called Src-B . While SRC, FYN and YES are ubiquitously expressed in mammals, the other members of the family show tissue-specific features. LYN and FGR are mainly expressed in the hematopoietic system ,2, whileDeciphering the role of SFKs in normal physiology, and in cancer, is highly complex. This is due to several factors, including functional redundancy between related members, inter-regulation between members, the complexity of the substrate network in SFKs, and their pattern of expression . Also, bSFKs switch from active forms  to inactive forms (phosphorylated at the C-terminus residue Y527) . The traAberrant activation of SFKs has been observed in various types of cancer, thus affecting their crucial roles in proliferation, survival, adhesion, chemotaxis, invasion and metastasis. It should be noted that, to date, no mutation of SFK genes has ever been identified in AML ,11,12. OIn the present review, we will summarize the current knowledge of SFK functions in acute myeloid leukemia and mastocytosis, with a focus on oncogenic FLT3 and KIT receptor signaling.Acute myeloid leukemia is characterized by the accumulation of immature hematopoietic cells in the bone marrow and blood. The disease is complex, with prognosis defined by various molecular mutations and cytogenetic profiles. The molecular heterogeneity of the disease, as well as clonal heterogeneity within patients, is a challenge when developing therapeutic strategies. Overall, AML remains a deadly disease, due to frequent relapse, and to the emergence of clones resistant to standard chemotherapy. Driver mutations in the FLT3 receptor occur in 25–30% of patients . The mosPoint mutations at codon D835, which mainly result in D to V amino-acid change, constitute another hotspot of FLT3 mutation. These account for 5% of AML cases, and, unlike ITD mutations, do not exhibit a clear influence on prognosis. Small chemical inhibitors of FLT3  are efficient reducers of the leukemic load in patients; however, during the following months rapid relapse is inevitable, due to the selection of cells that have developed resistance, or the selection of minor clones with no FLT3 mutations.Receptor tyrosine kinases such as FLT3 recruit intracellular signaling mediators directly, through binding to phosphorylated tyrosine residues which act as docking sites for SH2-containing proteins, or indirectly, through the formation of large protein complexes around adaptor/scaffold proteins, which constitute efficient signaling hubs. FYN, HCK, LCK, LYN, FGR and SRC interact, through their SH2 domains, with the docking sites Y589, Y591 and Y599, which are located in the juxtamembrane region of FLT3 ,21. FLT3KIT is another transmembrane receptor tyrosine kinase (RTK), structurally related to FLT3, CSF1R and PDGFR, with pleiotropic cellular functions, including cell differentiation, migration, survival and proliferation . In AML,Several SFKs are expressed in AML. Previous studies had suggested that expression of FGR and HCK in acute myeloid leukemia blasts was associated with early commitment and differentiation events in the monocytic and granulocytic lineages . MultiplInterestingly, analyses of gene expression in patient cohorts have produced evidences for the involvement of SFKs in AML. For instance, HCK is part of an LSC-enriched signature in AML . FurtherSFKs are not only expressed, but also activated, in most AML primary samples and cell lines regardless of their cytogenetic, molecular or cytological background ,29,33. LConsidering the importance of SFK signaling in solid cancers ,37, the Over the years, SFK inhibitors have been developed. Several have entered clinical trials, and some have obtained FDA approval for cancer treatment. These inhibitors are small chemical compounds which target the ATP-binding pocket of SFKs. Many tyrosine kinase inhibitors targeting SFKs have been used on cell lines, AML primary specimens, and in mouse models transplanted with human AML cells. The major limitation of these inhibitors has been their lack of selectivity: that is, they also inhibit many other kinases, including KIT and FLT3 wild-type and oncoproteins , in addi+CD38− AML cells in vitro, and reduced leukemic burden in mice engrafted with primary human AML cells [Several studies have shown that SFK inhibitors impair the leukemic phenotype of AML cells. The SFK inhibitor PP2 was shown to inhibit proliferation of AML cell lines , and to ML cells . InteresML cells . Taken tDespite caveats, SFK inhibitors have proved to be very useful tools for targeting these kinases in AML. The demonstration of SFK involvement, however, has required the use of independent but complementary technical approaches . This will be illustrated below using the example of AML harboring the FLT3-ITD oncoprotein.Inhibition of FLT3-ITD expression or the use of selective FLT3 inhibitors has demonstrated that SFK activation is dependent on FLT3 in all AML models studied, including mouse models, AML cell lines and patient samples.SFKs activate a wide range of proteins and signaling pathways. With regard to FLT3-ITD oncogenic signaling, the major contribution of SFKs has been highlighted in two essential pathways driving cell proliferation and cell survival. These pathways involve two key players: STAT5 and CDK6 .STAT5 is a crucial effector of FLT3-ITD oncogenic signaling . STAT5 iCDK4 and CDK6 are cyclin-dependent kinases responsible for progression of the G1 phase of the cell cycle. Two independent functional experimental approaches led to the conclusion that CDK6 is essential for proliferation in AML with the FLT3-ITD mutation. The first of these studies was a functional siRNA screen of all human kinases , while tIndependently, inhibition of STAT5 or CDK6 may be considered as novel strategies for AML management. However, SFK inhibition has the advantage of targeting both pathways. Furthermore, several SFK inhibitors also hit essential hematopoietic receptors such as FLT3 or KIT, which might prove of additional interest in attempts to eradicate leukemic cells.In hematopoiesis, following the binding of KIT ligand, stem cell factor (SCF), KIT dimerizes and initiates the subsequent activation of downstream signaling pathways, which have been extensively studied in different cell types. Four main pathways have been described: the PI3-Kinase/AKT, RAS-ERK, PLC-γ and SFK signal transduction pathways . SCF binD816V, hijack the wild-type signaling circuit of KIT to promote their tumorigenic potential [WT is highly transient [D816V, and are involved in its oncogenic activity [Interestingly, it has been shown that oncogenic forms of KIT, such as KITotential ,61,62. Notential ,64. For ransient ,67,68,69ransient ,70. In aactivity .D816V and KITN822K [D816V activates SFKs, whereas KITN822K activates ERK, a pathway that is downregulated by KITD816V (and KITWT) [D816V is associated with a poorer prognosis than KITN822K [Interestingly, two of the main KIT mutants identified in CBF-AML, KITKITN822K , generatd KITWT) ,70. AlthKITN822K ,73, the The role of SFKs downstream of KIT in AML remains insufficiently understood. Since KIT is highly expressed in most AML cases, and is a recognized therapeutic target, it is of interest that some of the SFK inhibitors in clinical use, e.g., midostaurin, dasatinib, bosutinib, and ponatinib, also target KIT.Mastocytosis is a rare heterogeneous disorder associated with abnormal clonal expansion and accumulation of mast cells in various tissues. There are three major forms of mastocytosis as defined by the World Health Organization (WHO): mast cell sarcoma, cutaneous mastocytosis (CM), and systemic mastocytosis (SM) ,75. ThisMast cells are known to be essential for host defence responses, but also to play a role in allergy and anaphylaxis. They express a variety of receptors, including FcεRI and KIT, in order to respond to endogenous and exogenous stimuli . The higD816V, induces a structural change in the catalytic domain, resulting in a permanent structural active state, and thereby the constitutive activation of the receptor tyrosine kinase [Mast cells are the only terminally differentiated hematopoietic-derived cells to express the stem and progenitor cell marker, the KIT receptor. The predominant genetic alteration identified in more than 80% of adult patients with mastocytosis is a somatic point mutation in the c-KIT gene, leading to an amino-acid change at position 816. This mutant, KITe kinase ,80.−/− mouse models, the role of LYN in bone marrow-derived mast cell (BMMC) in vitro proliferation, degranulation (histamine release) and calcium mobilization has been studied by various groups. However, their findings were, and remain, controversial, with reports of LYN promoting, decreasing or having no effect on the same function and signaling transduction pathways [As described in pathways ,90,91,92−/− mouse model have identified HCK as an important factor for optimal mast cell proliferation, downstream of KITWT [Numerous hypotheses, such as the use of different mouse model backgrounds, or the number of passages at which BMMCs were used , have beof KITWT .WT in mast cells are similar to those described previously in normal hematopoietic progenitor cells ; it was less frequently activated in indolent systemic mastocytosis (ISM) patient samples . HCK wasreceptor . The dowreceptor . Interesll death ,85. AccoWild-type KIT receptor, by itself, is unable to induce degranulation or cytokine production, but can enhance these cellular responses, mediated by the high affinity surface receptor for IgE, FcεRI .To date four SFKs have been functionally involved downstream of FcεRI: LYN, FYN, HCK and FGR (for review: ); they aLYN was the first SFK found to be associated with FcεRI, and to be activated following receptor aggregation . LYN is −/− BMMCs) [FYN is also expressed in BMMCs, and is activated following its interaction with engaged FcεRI, which then induces its interaction with GAB2, promoting GAB2 phosphorylation and subsequent activation of the PI3Kinase/AKT transduction pathway . As an u− BMMCs) ,87. FYN − BMMCs) . STAT5 i−/− BMMCs have revealed that HCK is a positive mediator of FcεRI-mediated degranulation, and is an important activator of both JNK and p38 MAP-Kinases and the PI3Kinase/AKT transduction pathways, and, surprisingly, of SYK [HCK is expressed in BMMCs at lower levels than LYN and FYN , and doe, of SYK .Finally, the most recent SFK to have been linked to FcεRI is FGR . FGR int-/- BMMCs [−/− BMMCs [The assembly of FcεRI subunits then initiates complex SFK signaling transduction, activating LYN, FYN, HCK and FGR. Their individual impact is not redundant as they activate different downstream signaling transduction pathway. These four SFKs are nevertheless interconnected, since their activity is dependent on other members of the family, as demonstrated by the study of mouse knock-out mast cells: FYN kinase activity is upregulated in lyn/- BMMCs , and LYN/− BMMCs .KIT and FcεRI receptor signaling orchestrate mast cells responses. The addition of KIT-ligand to human or murine mast cells potentiates FcεRI-mediated degranulation ,107,108.D816V activity does not affect LYN or BTK constitutive activation in AdvSM patient samples, but their down-regulation by RNA interference decreases survival in KITD816V-expressing cell lines [As stated above, the inhibition of KITll lines . This obll lines .D816V activity, Lortholary and colleagues [Other drugs that target SFKs in addition to other protein tyrosine kinases have been developed ,112. Unflleagues  observedlleagues .SFKs are therapeutic targets of interest both in AML and mastocytosis. Yet, some challenges remain in order to fully decipher their role(s) in these contexts, and then to translate this knowledge to the clinic.In AML, SFK activation is a common feature of most samples, despite the diversity of this disease. There is evidence that some SFK inhibitors also impair LIC activity, which is a promising property. Indeed, relapse and resistance to current therapies are still the main challenge in AML, as leukemic cells are extremely plastic and they inevitably escape single agent therapy. An important strategy is therefore the use of combination regimens. The prevalent and empirical approach is to combine SFK inhibitors with established chemotherapy or another existing targeted therapy in AML, and such clinical trials are currently ongoing. More innovative and rational treatments based on experimental evidence may shortly come up from dedicated functional screens of drug libraries or genetic tools targeting the whole genome.Mastocytosis is due to an excess number of hyperactivated mast cells in various tissues. Indeed, mast cells release numerous mediators from internal granules, an event responsible for the symptoms. SFK-targeting drugs impair mast cell degranulation, as a result of FcεRI signaling blockade. Here, the combination of drugs hitting both the proliferation and survival functions controlled by mutant KIT receptor, and FcεRI signaling using SFKinhibitors, is also a promising objective of research.


Prognosis in pediatric patients with refractory/relapsed acute myeloid leukemia (AML) is grim, and there is no standard treatment for such patients. Combined treatment with intensive chemotherapy and gemtuzumab ozogamicin (GO), a monoclonal anti-CD33 antibody conjugated with calicheamicin, is useful as reinduction therapy in refractory/relapsed AML. Here, we describe three cases of pediatric refractory/relapsed AML that were successfully managed with FLAG-IDA , with or without GO, as reinduction therapy before a KIR-ligand-mismatched cord blood transplant. This strategy relies on the fact that killer cell immunoglobulin-like receptors (KIR) on cord blood natural killer (NK) cells recognize human leukocyte antigen (HLA) class I alleles, and that donor KIR-ligand incompatibility may be associated with lower incidence of relapse and improved survival in AML, as cells that lack these inhibitory HLA ligands can activate NK cells. All three patients are currently alive and have been disease-free for 24–65 months, although one patient developed severe sinusoidal obstructive syndrome (SOS). Thus, our strategy can result in excellent outcomes in pediatric patients with refractory/relapsed AML. Outcomes in children with refractory/relapsed acute myeloid leukemia (AML) are extremely poor . Fludara2 fludarabine and 2 g/m2 cytarabine on days 2–6, 10 mg/m2 idarubicin on day 6, and 300 µg/m2 G-CSF on days 1–6. GO was administered at a dose of 3 mg/m2 on day 7 of treatment, and its use was approved by the institutional review board of the Showa University Fujigaoka Hospital, Japan. All cases achieved CR after reinduction therapy (2), fludarabine (125 mg/m2), and TBI (2 Gy), as this was her 2nd HSCT. Anti-thymocyte globulin (ATG) was not used in any of the patients, and tacrolimus (0.02 mg/kg/day) and short-term methotrexate were used for prophylaxis against graft-versus-host disease (GVHD). Ursodeoxycholic acid (UDCA) and low-molecular-weight heparin (LMWH) were administered for prophylaxis against sinusoidal obstruction syndrome (SOS) in case 1, while cases 2 and 3 received prophylactic recombinant human soluble thrombomodulin , in addition to UDCA and LMWH [Between April 2011 and December 2015, one male and two female pediatric patients with relapsed or refractory AML were treated with one course of FLAG-IDA, with or without GO, after which they underwent KIR-CBT. Patient characteristics are listed in  therapy  and prom therapy . KIR-lig2), is a feasible induction therapy option in younger patients with primary AML [Prognosis in pediatric patients with refractory/relapsed AML is poor at best and known prognostic factors for poor survival include early relapse, undergoing HSCT during 1 CR, age of >10 years at relapse, and adverse cytogenetics . A Europmary AML , 4. All mary AML . Ruggerimary AML . In a stmary AML . In contmary AML . It is pmary AML . Rubnitzmary AML . As thesmary AML , while Lmary AML . Those nIn conclusion, the FLAG-IDA reinduction therapy described here, with or without GO, when used before KIR-CBT, appears to be a feasible treatment strategy in pediatric patients with refractory/relapsed AML. However, definitive conclusions about the efficacy of this approach cannot be made because of the very small number of patients included in this study. Therefore, additional prospective studies are required to evaluate the same.


Here, we investigate the differential impact of corticosteroid-mediated increase in ICaL on action potentials (AP), ion currents, intracellular Ca2+ handling and contractility in endo- and epicardial myocytes of the rat left ventricle. Dexamethasone led to a similar increase in ICaL in endocardial and epicardial myocytes, while the K+ currents Ito and IK were unaffected. However, AP duration (APD) and AP-induced Ca2+ influx (QCa) significantly increased exclusively in epicardial myocytes, thus abrogating the normal differences between the groups. Dexamethasone increased Ca2+ transients, contractility and SERCA activity in both regions, the latter possibly due to a decrease in total phospholamban (PLB) and an increase PLBpThr17. These results suggest that corticosteroids are powerful modulators of ICaL, Ca2+ transients and contractility in both endo- and epicardial myocytes, while APD and QCa are increased in epicardial myocytes only. This indicates that increased ICaL and SERCA activity rather than QCa are the primary drivers of contractility by adrenocorticoids.Regional differences in ion channel activity in the heart control the sequence of repolarization and may contribute to differences in contraction. Corticosteroids such as aldosterone or corticosterone increase the L-type Ca Aldosterone thereby controls extracellular fluid volume and arterial blood pressure1. Beyond its action on Na+-absorbing epithelia, the effects of MR activation on various non-epithelial targets, such as the heart, are increasingly recognized. However, its mechanisms of action on these non-classical targets are far less understood. Clinical trials provide convincing evidence that MR antagonistic therapy increases survival in patients with heart failure, by, among others, decreasing the rate of fatal arrhythmias3, which are facilitated by electrical remodeling of the myocardium4. In line with this observation, a number of studies have demonstrated that MR activation modulates ionic currents and transporters in ventricular cardiomyocytes, including the fast Na+ current5, the hyperpolarization-activated and cyclic-nucleotide gated current6, the ryanodine receptor7, and the L-type Ca2+ current (ICaL)8. The latter is of particular importance as alterations in ICaL not only affect cardiac electrophysiology and contractility directly, but also serve as a first step in chronic electrical and mechanical remodeling of the heart10. Consequently, mice overexpressing the MR show electrical abnormalities, arrhythmia and cardiac hypertrophy11.Aldosterone, a corticosteroid hormone of the adrenal gland and a key player in the renin–angiotensin–aldosterone cascade, has a pivotal role in maintaining total body Na12. Although differences in a number of ionic currents contribute to the heterogeneity in APD among the ventricle14, differences in the magnitude of the transient outward K+ current (Ito) between endo- and epicardial layers of the ventricle are the most prominent. In rodents such as mice and rats, these differences in Ito are mainly responsible for the gradient in APD among the left ventricular free wall16, while in larger mammals such as dog or humans, Ito magnitude exerts less influence on total APD, but rather controls the membrane voltage level during the early part of the plateau phase19. Since the AP constitutes the driving force for the ionic currents flowing during the AP, differences in AP waveform directly affect ion fluxes across the membrane. This is particularly important for the L-type Ca2+ current, as the amount of Ca2+ entering the myocytes during an AP controls excitation–contraction–coupling. For example, despite a similar ICaL magnitude in endo- and epicardial myocytes, the longer AP in endocardial myocytes leads to an increased AP-induced Ca2+ influx20. Differences in duration and shape of the AP, therefore, not only control the sequence of repolarization but also affect Ca2+ influx, intracellular Ca2+ cycling, contractility and cardiac remodeling23. The particular relevance of the AP shape is highlighted by studies, in which epicardial myocytes displayed increases in Ca2+ influx, Ca2+ transients and contractility, when clamped on an endocardial AP and vice versa24. Moreover, alterations in AP waveform in ferret myocytes caused desynchronized SR Ca2+ release which is typically observed in myocytes originating from failing hearts25. Although the magnitude of Ito is the major parameter responsible for differences in AP shape and duration among different regions of the ventricular wall, it is important to note that the AP waveform of individual myocytes is a function of the cell’s magnitude of both, Ito and ICaL. In experiments, in which Ito, ICaL and APD were measured in the same cells, Gomez and coworkers showed that early repolarization of the rat AP (APD20) neither correlated with the magnitude of ICaL nor with Ito alone, but only with both currents together26. This suggests that changes not only in the magnitude of Ito, but also in ICaL might substantially affect AP waveform and in turn Ca2+ influx, Ca2+ transients and contractility.In the normal heart, repolarization is a well-coordinated process controlled by the sequence of excitation and regional differences in action potential duration (APD) among the ventricles. In fact, the regions excited early, such as endocardial or septal parts of the left ventricle, repolarize last due to longer action potentials (AP) and vice versaCaL affects AP shape, Ca2+ influx, Ca2+ transient and contractility differently in endo- and epicardial myocytes. To address this question, we investigated the influence of mineralocorticoid receptor activation on ICaL, AP-induced Ca2+ influx, contractility, Ca2+ transient and SR Ca2+ handling in endo- and epicardial myocytes isolated from the rat left ventricle. We show that MR activation substantially increases contractility to a similar extent in both endo- and epicardial myocytes but modulates intracellular Ca2+ handling differently.In the present study, we therefore hypothesized that the corticosteroid induced increase in I2+ current (ICaL) via activation of the mineralocorticoid receptor (MR)27. In the present study, we used dexamethasone as corticosteroid to stimulate ICaL. Moreover, in a previous study27, insulin was part of the primary culture conditions and we later learned that it is a prerequisite for ICaL regulation by corticosteroids: Fig. CaL obtained from a control myocyte, a myocyte incubated with 1 µM dexamethasone, a myocyte incubated with 100 nM insulin and a myocyte incubated with the combination of both dexamethasone and insulin. 24 h incubation with dexamethasone substantially increased ICaL only in the presence of insulin. The current–voltage (I–V) relations shown in Fig. CaL at 0 mV by 46% from 7.0 ± 0.5 pApF−1 (n = 21) to 10.2 ± 0.8 pApF−1  while in the presence of dexamethasone  or insulin  alone, no significant difference was observed. For that reason we combined 1 µM dexamethasone with 100 nM insulin (DI) in all following experiments. To address the question whether DI increases ICaL via the MR or the glucocorticoid receptor (GR), we incubated myocytes with DI alone and with DI and the MR antagonist spironolactone (10 µM). 24 h incubation with DI substantially increased ICaL, while in the presence of spironolactone, ICaL remained at the level of the control myocytes  to 11.1 ± 0.6 pApF−1 . In the presence of spironolactone, DI did not significantly affect ICaL, indicating that dexamethasone, like aldosterone or corticosterone27, increases ICaL via the MR.Incubation of isolated left ventricular cardiomyocytes for 24 h with corticosteroids, such as the mineralocorticoid aldosterone or the glucocorticoid corticosterone, has been shown to increase the L-type CaCaL, we isolated endo- and epicardial myocytes from the left ventricular free wall and investigated ICaL after 24 h incubation with DI. Figure CaL recorded from endo (left panel) and epicardial (right panel) myocytes. Compared to control, DI increased ICaL at 0 mV in endocardial and in epicardial myocytes to a similar extent by 45%  and 49% , respectively. We also investigated the effect of DI on repolarizing K+ currents. The transient outward K+ current (Ito) displayed the typical gradient among the left ventricular free wall with much larger currents in epi-compared to endocardial myocytes. However, in both endo- and epicardial myocytes, Ito magnitude was not affected by 24 h incubation with DI , we used the current at the end of a 600 ms voltage pulse15. Figure K.To investigate regional differences in the effects of corticosteroids on I90) increased from 90.2 ± 11.4 to 220.2 ± 31.4 ms  from endo- and epicardial myocytes. Figure CaL current is not a rectangular voltage pulse such the ones used in Figs. 2+ influx. We therefore recorded APs from myocytes and subsequently used their own individual AP as voltage template for the following voltage clamp experiments, in which we clamped the membrane voltage of each myocyte on its own AP in the absence and in the presence of 100 µM Cd2+ to inhibit ICaL. The resulting current is a good estimate of the AP-induced Ca2+ current20. Figure 2+ currents obtained from individual myocytes of endo- and epicardial origin recorded under control conditions (blue) and after incubation with DI (green). Incubation with DI increased the peak AP-induced Ca2+ current in endo- and epicardial myocytes to a similar extent , i.e. the total amount of Ca2+, entering the myocyte via L-type channels during an AP. As we have published previously20, the long endocardial AP leads to a significantly larger QCa in endo-compared to epicardial myocytes under control conditions. Incubation with DI dramatically increased QCa by 301%  in epicardial myocytes, while in endocardial myocytes the increase was much smaller  and did not reach statistical significance  obtained from an endo- and an epicardial myocyte under control conditions (blue) and after 24 h incubation with DI (green), while Fig. Ca in endocardial cardiomyocytes, the amplitude of Ca2+ transients  as well as fractional unloaded sarcomere shortening  were similar in endocardial and epicardial myocytes after 24 h of incubation under control conditions  as well as in epicardial cardiomyocytes . Accordingly, unloaded fractional shortening increased by 74%  in endocardial cells and by 45%  in epicardial myocytes  and after incubation with DI (green). Since caffeine locks the ryanodine-receptor in an open state, Ca2+ release from the SR is maximal28. Accordingly, the Ca2+ transients were substantially larger than those observed in response to pacing. SR Ca2+ content was assessed as the amplitude of the Ca2+ transients and was similar in endo- and epicardial myocytes under control conditions. Interestingly, DI did not significantly alter SR Ca2+ content  leaving residual Ca2+ transients arising from Ca2+ influx via L-type Ca2+ channels only. Figure 2+ transients stimulated by 1 Hz pacing. The arrow indicates the application of thapsigargin to the bath solution. Within 3–5 min, Ca2+ transients had decreased to a residual amplitude reflecting Ca2+ influx from the extracellular space only. Figure 2+ transients before and after the application of thapsigargin thus equaling the amount of Ca2+ released from the SR. In both, endo- and epicardial myocytes, DI substantially increased SR Ca2+ release. Since the total amount of Ca2+ in the SR was not affected  and Ca2+ reuptake into the SR via SERCA should be increased in myocytes treated with DI. We therefore assessed time and rate constants of the decline of Ca2+ transients during regular pacing and in response to application of caffeine. In DI treated myocytes the time constant of the Ca2+ transient decay was accelerated in both endo- and epicardial myocytes  was also substantially accelerated by DI treatment.In light of increased Cates Fig.  indicatiFinally, to address mechanisms underlying the increased SERCA transport rate, we performed western blot experiments in cardiomyocytes incubated for 24 h under control conditions and after treatment with DI to quantify SERCA and phospholamban expression as well as phospholamban phosphorylation. Since SERCA activity was similar in endo- and epicardial myocytes of both, control and DI groups, and in order to increase the total protein yield, we used isolated myocytes of the whole left ventricle for western blot experiments. Figure CaL to a similar extent in endo- and epicardial myocytes, demonstrating that, independent of regional origin, ICaL is not only similar in magnitude, but is also identically affected by MR agonists. Although treatment with DI led to a much larger increase in APD and AP-induced Ca2+ influx in epicardial myocytes, the increase in intracellular Ca2+ transient and, hence, contractility was similar in both regions. Moreover, SR Ca2+ content was similar in both regions and not affected by DI treatment. We discovered that in both regions the fractional release of Ca2+ from the SR was increased by DI treatment. Our results suggest that the increase in ICaL density rather than APD or AP-induced Ca2+ influx controls the increase in Ca2+ transient and contractility observed by DI treatment.In the present study, MR activation increased I20. Specifically, APD was substantially shorter in epicardial than in endocardial myocytes. This difference is attributable at least to a large extent to the larger epicardial Ito current density, while other ionic currents, such as IK and ICaL, were similar in both regions. Since the AP waveform constitutes the voltage driving force for ionic currents including ICaL, the longer AP in endocardial myocytes led to an increased AP-induced Ca2+ influx in endocardial compared to epicardial myocytes, despite of a similar current density of ICaL in both regions. This is in line with previous observations30 and underlines the importance of AP shape for control of the AP-induced Ca2+ influx. Moreover, all recordings in the present study were performed after at least 24 h incubation, hence, our data show that the regional differences in APD, underlying ionic currents and AP-induced Ca2+ influx remain well preserved even after a prolonged (24–36 h) period of primary culture.In the present study, we confirm regional differences in AP shape and duration as well as in underlying ionic currents that we and others have previously described for the left ventricular free wall of the rat2+ influx in endocardial myocytes, the intracellular Ca2+ transient as well as unloaded sarcomere shortening were similar in myocytes obtained from both regions. This fits well to previous observations in canine32 and rabbit33 left ventricle, while in mouse left ventricle a larger Ca2+ transient was found in endocardial myocytes34. For the rat left ventricle, conflicting results have been reported. Fowler et al. showed larger Ca2+ transients in endocardial myocytes probably evoked by an increased SR Ca2+ content35. However, they did not address myocyte contractility. On the other hand, Cazorla et al. reported similar Ca2+ transient amplitudes36 and Smail et al. also reported similar Ca2+ transient amplitudes and myocyte shortening in endo- and epicardial myocytes37. We also found no significant difference in Ca2+ transient amplitude or sarcomere shortening. Moreover, in our hands SR Ca2+ content was similar in endo- and epicardial myocytes, suggesting that the larger AP-induced Ca2+ influx in endocardial myocytes does not result in an increased SR Ca2+ filling state. In a state of intracellular Ca2+ homeostasis, a larger Ca2+ influx from the extracellular space into the cytoplasm during each AP must be matched by an increased Ca2+ extrusion into the extracellular space, carried predominantly by the NCX38. Consistently, we found a trend towards a shorter time constant of NCX Ca2+ removal in endocardial compared to epicardial myocytes  increased AP-induced Ca2+ influx41. One would therefore expect that an acute delay in early repolarization caused by an increase in ICaL should even further increase the AP-induced Ca2+ influx, since not only the driving force  changes, but also the Ca2+ conductance of the membrane. Indeed, the increase in ICaL we observed in response to MR activation substantially delayed early repolarization and increased APD, predominantly in epicardial myocytes, to such an extent that the endo-epicardial differences in APD disappeared. As a consequence, AP induced Ca2+-influx increased substantially in epicardial and to a lesser (but not significant) extent in endocardial myocytes. The relatively small increase of the AP induced Ca2+-influx in endocardial myocytes is consistent with their per se longer AP and delayed early repolarization, which leaves less room for an increase in APD upon an increase in ICaL.It is well established that MR activation for > 18–24 h increases I2+ transient in both endo- and epicardial myocytes. A prolonged AP with a resulting increase in sarcolemmal Ca2+ influx might alter several factors contributing to the intracellular Ca2+ transient: an increase in Ca2+ influx can trigger an increased release from the SR and might also increase the SR Ca2+ content which in turn increases SR Ca2+ release. Moreover, fractional release of Ca2+ from the SR is controlled by two mechanisms, the SR Ca2+ content and the magnitude of the trigger Ca2+, i.e. the AP-induced Ca2+ influx42. In the present study, we found no increase in SR Ca2+ content despite a substantial increase in AP-induced Ca2+ influx and in SERCA activity. The increase in amplitude of the Ca2+ transient can be explained by an increase in fractional release from the SR and (to a lesser extent) by an increase in trans-sarcolemmal Ca2+ influx. This finding is supported by previous observations. Trafford et al. showed that an increase in Ca2+ influx can substantially increase Ca2+ release from the SR (and contractility) in the absence of effects on SR Ca2+ content44. In animal models of heart failure with an increase in APD, Sah41 and Kaprielian45 observed an increased Ca2+ release from the SR in the absence of an increased SR Ca2+ content. In our study, fractional Ca2+ release averaged ~ 40% under control conditions which is similar to the ~ 50% observed by Picht et al.46. In cardiomyocytes from mice overexpressing the MR or after 48 h of incubation with aldosterone, SR Ca2+ content was also unaltered compared to control myocytes7. Since Bassani et al.42 found only a 4% increase in SR Ca2+ content in response to a switch to high loading condition, one could speculate that already under control conditions, SR Ca2+ content might be nearly maximal. Moreover, Bode et al.47 could show that in rat cardiomyocytes, SR Ca2+ content only weakly depends on SERCA activity, when the SR Ca2+ content is high. This was explained by an increase in SR Ca2+ leak. Interestingly, aldosterone has been shown to increase SR Ca2+ leak by downregulation of FKBP12 and 12.6 expression, thereby further limiting a potential increase in SR Ca2+ load7. SERCA activity is in addition modulated by SUMOylation49. SUMOylation of SERCA by SUMO1 is decreased in heart failure and contributes to decreased SERCA activity under this condition. Moreover, β-arrestin-2 enhances SERCA SUMOylation49. Interestingly, glucocorticoids decrease β-arrestin-2 expression at least in human lung adenocarcinoma cells50, could thus indirectly decrease SUMOylation of SERCA and thereby reduce SERCA activity. Taken together, modulation of ICaL density via the MR appears as a potent regulator of intracellular Ca2+ transient magnitude and contractility in both, endo- and epicardial myocytes.MR activation led to a substantial increase in the intracellular Ca51. After induction of deep anesthesia by intraperitoneal injection of thiopental-sodium (100 mg kg−1 body mass), the heart was quickly excised and placed into cold (4 °C) Tyrode’s solution. Subsequently, the aorta was retrogradely perfused for 5 min with modified Tyrode’s solution containing 4.5 mM Ca2+ and 5 mM EGTA (~ 1 μM free Ca2+ concentration) supplemented with 1 μM insulin . The perfusion was continued for 19 min, recirculating 25 ml of the same solution containing collagenase  and protease . Then, the heart was perfused for another 5 min with storage solution8 containing 100 μM Ca2+. Using fine forceps, tissue portions of the subendocardial  and subepicardial  layers were taken and placed in separate cell culture dishes containing the same solution at 37 °C. Tissue pieces were minced and gently agitated to obtain single cardiomyocytes. Myocytes were stepwise adapted to physiological Ca2+ levels, transferred to cell culture dishes containing storage solution supplemented with 1 g l−1 BSA, 100 IU ml−1 penicillin and 0.1 mg ml−1 streptomycin and stored at 37 °C in a water-saturated atmosphere containing 5% CO2. For incubation with dexamethasone and insulin, appropriate amounts of stock solutions containing dexamethasone (dissolved in 100% ethanol) and insulin were added to the respective cell culture dishes; the corresponding amount of ethanol was added to control groups, the final concentration of ethanol was ~ 0.02%. Isolated cardiomyocytes were used for experiments for up to 36 h. Only quiescent single rod-shaped cells with clear cross striations were used for experiments. All experiments were performed in accordance with relevant guidelines and regulations and all experimental protocols were approved by the Regierung von Mittelfranken, license No: 621–2,531.32–11/05.Cardiomyocytes were isolated from the left ventricular free wall of female Wistar rats (~ 220 g) as described previously52. Myocardial cells were transferred into an elongated chamber (2.5 × 20 mm), mounted on the stage of an inverted microscope  and initially superfused with control solution. All experiments were performed at room temperature (22–25 °C). Patch pipettes were pulled from borosilicate glass  using a P-97 Puller . Pipette resistance (RPip) was 1.5—5 MΩ. Currents were recorded using an EPC-10 amplifier , controlled by PULSE-Software (HEKA Elektronik). Membrane voltage (Vm) and APs were recorded in the zero current-clamp mode and ionic currents in the voltage-clamp mode. For AP voltage-clamp recordings, APs were recorded at the beginning of the experiments and used as a voltage template in the voltage-clamp mode of the amplifier53. Membrane capacitance (Cm) and series resistance (Rs) were calculated using the automated capacitance compensation procedure of the EPC-10 amplifier. Series resistance was in the range of ~ 5 MΩ, was not allowed to exceed 10 MΩ and was compensated by 85%. The reference electrode of the amplifier headstage was bathed in pipette solution in a separate chamber and was connected to the bath solution via an agar–agar bridge filled with pipette solution. Pipette potential (VPip) and Vm were corrected for liquid junction potentials at the bridge-bath junction. Whole-cell currents were low-pass filtered at 1 kHz and sampled at 5 kHz. Action potentials were sampled at 5 kHz.The ruptured-patch whole-cell configuration was used as described previouslyCaL, myocytes were clamped for 600 ms from the holding potential of − 90 mV to test potentials between − 60 mV and + 70 mV in steps of 10 mV. Na+ currents were inactivated by a prepulse of 70 ms to − 50 mV. Basic cycle length was 3,000 ms. ICaL was quantified by subtracting the current at the end of the test pulse from the peak current.54 To elicit outward K+ currents, myocytes were clamped for 600 ms from the holding potential of − 90 mV to test potentials between 60 mV and − 80 mV in steps of − 20 mV. Na+ currents were inactivated by a prepulse of 20 ms to − 50 mV. Basic cycle length was 3,000 ms. Ito was quantified by subtracting the current at the end of the test pulse from the peak current. IK was defined as the current at the end of the voltage pulse.To assess I2+ epifluorescence was recorded as previously described56. Cells were incubated in modified Tyrode’s solution with Fura-2-AM (4 μmol/l). Cells were transferred into a chamber (self-manufactured) mounted on an inverted microscope . Ca2+ transients were recorded during field stimulation . Cells were alternatively excited at 340 and 380 nm . The F340/F380 ratio was used as an index of cytosolic Ca2+ concentration. In some experiments, caffeine or thapsigargin were washed in. For all Ca2+ fluorescence experiments, cells were paced for 1 min at 1 Hz before starting the measurements to ensure that Ca2+ balance was at steady state.Ca2O) were added. Handled on ice at all times, samples were mechanically homogenized for 20 s, sonicated for 3 × 5 s and then centrifuged at 13,000 g and 4 °C for 10 min. The supernatant was used for further studies. To ensure equal protein loading, protein concentration was measured using the BCA Protein Assay Reagent Kit for microplate assay . BSA in concentrations between 25 and 2000 µg/ml in TNE buffer was used as standard.Isolated cardiomyocytes were incubated with DI for 24 h while myocytes isolated from the same heart were incubated for 24 h with vehicle and served as paired control. Myocytes were pelleted by centrifugation and dispensed in 1 ml TNE buffer. 40 µl protease inhibitor, 40 µl phosphatase inhibitor, 30 µl triton X-100 (10%), 5 µl PMSF (200 mM in 100% EtOH) and 20 µl sodium deoxycholate (12.5% in H57.Samples were prepared for electrophoresis by adding Roti-Load 1 (4 × concentrated)  at 1:4 to respective identical amounts of protein. Samples were heated to 95 °C for 5 min and then loaded pairwise (DI and the corresponding control) into the wells of 8% or 12% SDS polyacrylamide gels, separated by electrophoresis and blotted to PVDF membranes, which were blocked in TBST solution containing 1% dry milk and probed for SERCA and phospholamban. Primary antibodies were: goat SERCA2 (C-20): sc-8094 , mouse phospholamban: A010-14 , rabbit phospholamban pSer16: A010-12 (Badrilla Ltd.), rabbit phospholamban pThr17: A010-13 (Badrilla Ltd.). Secondary antibodies were: goat anti-rabbit , rabbit anti-goat , goat anti-mouse . Blots were developed using the Super Signal West Femto Maximum Sensitivity Substrate . Chemiluminescence was acquired using a STELLA imager with the XStella and AIDA image analyzer software . Image saturation was prevented by adjusting exposure times of the camera. Using serial dilutions we ensured for each antibody that the applied amounts of protein were within the linear detection range of the imaging system. The amount of total protein was assessed using Ponceau S staining2PO4 0.33, MgCl2 1, HEPES 10, CaCl2 4.5, EGTA 5, titrated to pH 7.30 using NaOH. The same solution without EGTA and with 2 mM Ca2+ was used as bath solution for the patch-clamp and fluorescence imaging experiments. For cell digestion, collagenase  and protease  were added to modified Tyrode’s solution (10–6 M Ca2+). Storage solution contained (in mM): NaCl 130, NaH2PO4 0.4, NaHCO3 5.8, MgCl2 0.5, CaCl2 1, KCl 5.4, glucose 22, and HEPES 25, titrated to pH 7.40 with NaOH in the presence of 5% CO2 and supplemented with 1 mg ml−1 BSA. For recording action potentials and K+-currents, the pipette solution contained (in mM): glutamic acid 120, KCl 10, MgCl2 4, EGTA 10, HEPES 10, and Na2ATP 2, pH 7.20 with KOH. For recording Ca2+ currents, the pipette solution contained (in mM): CsCl 130, MgCl2 5, EGTA 10, HEPES 10, Na2ATP 2, pH 7.20 with CsOH.For the isolation of myocytes, modified Tyrode’s solution contained (in mM): NaCl 138, KCl 4, Glucose 10, NaHFor protein extraction and western blots, TNE buffer contained (in mM): Tris 20, NaCl 150, EDTA 1, pH = 7.40 and was completed with protease inhibitor  and PMSF (1 mM) just before starting protein extraction. SDS sample buffer was the mix from the homogenizing buffer and 4xRoti-Load . TBST solution contained (in mM): Tris 50, NaCl 150, 0.05% Tween-20, pH 7.6 with HCl.54. Ca2+ epifluorescence data were analyzed using Ionwizard 5.0  and Microsoft Excel. Quantitative densitometric analysis of western blots was performed using ImageJ software. The intensity of specific bands was determined after background subtraction and normalized to the total protein content per lane as quantified by densitometric analysis of the corresponding Ponceau stains58.Patch clamp data were analyzed using the PULSE-FIT software , IGOR Pro , and Microsoft Excel  as described previouslyt test when two groups were compared or one-way ANOVA followed by Newman-Keuls test  when more than two groups were compared using Prism 5 . p < 0.05 was considered statistically significant.Data are given as mean ± SEM. Statistical significance was evaluated by paired or unpaired Student's Supplementary information 1


Here, we report that TRIC-A–mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A–dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release–activated Ca2+ channel 1 (Orai1) within ER–plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca Trimeric intracellular cation (TRIC) channels have been proposed to modulate calcium release from the endoplasmic reticulum (ER) and to determine oscillatory calcium signals. This study shows that upon depletion of ER calcium, TRIC-A coclusters and interferes with STIM1-Orai1 coupling within ER-plasma membrane junctions. Both TRIC isoforms (TRIC-A and TRIC-B), also known as transmembrane protein 38A (TMEM38A) and TMEM38B, respectively, are ER-resident proteins with monovalent cation permeability ER). STIM1 is an ER-resident Ca2+ sensor for [Ca2+]ER with its N-terminal EF-hand bound to [Ca2+]ER. Following [Ca2+]ER depletion, Ca2+ dissociates from the EF-hand and causes conformational changes in STIM1 that result in oligomerization and translocation of the protein to ER–plasma membrane (PM) junctions [2+ entry via Orai1 causes sustained elevation of intracellular Ca2+ ([Ca2+]i) in cells where [Ca2+]ER depletion is substantial, i.e., those stimulated with high [agonist]. Alternatively, in cells stimulated with relatively low [agonist], SOCE drives sustained Ca2+ oscillations, which are variable in frequency and linked specifically to downstream signaling pathways ER sensing cation channel involved in facilitating [Ca2+]ER release and regulating intracellular K+/Ca2+ homeostasis. How the intracellular channels modulate oscillatory Ca2+ signals is as yet incompletely understood. Here, we explored the function of TRIC-A by heterologous expression in human embryonic kidney 293 (HEK293) cells engineered to reconstitute muscle-type oscillatory Ca2+ cycling. This system enables investigation of TRIC-A function in the absence of endogenous background expression of this isoform. We provide evidence that TRIC-A modulates SOCE by affecting STIM1/Orai1 complex assembly and function, as well as ER Ca2+ store refilling. We show that TRIC-A channels cocluster with the STIM1/Orai1 Ca2+ signaling complex within ER–PM junctions upon ER Ca2+ store depletion. Coassembly of TRIC-A with STIM1/Orai1 attenuates STIM1/Orai1 interaction and SOCE-driven Ca2+ oscillations. Hence, we propose a novel, to our knowledge, function for TRIC-A as a modulator of SOCE and oscillatory Ca2+ signals. Aberrant SOCE and cellular Ca2+ handling could underlie the muscle disorders associated with loss of TRIC-A.A number of previous studies have identified TRIC-A as a unique, muscle-specific o), ischemia/reperfusion, digitalis toxicity, and independently of membrane depolarization [2+]o increases the steady-state ER Ca2+ content in HEK293_RyR2 cells, thereby promoting the discharge of the Ca2+ stores via RyR2, which are at the same time governed by local Ca2+ levels phenyl]-4-methylthiadiazole-5-carboxamide  [2+ influx pathway for refilling ER Ca2+ stores and initiating Ca2+ discharge during the oscillatory cycle.HEK293 cells stably expressing RyR2 (HEK293_RyR2 cells) offer an established model of relatively simple molecular composition to mimic the cardiac phenotype for studying RyR2-induced spontaneous Ca (SOICR) . These orization . Raising+ levels . We firsgenously . In S1B o 1.0 mM . As expecontrols . This frls (2+]o . Moreove]o (2+]o . To test 10 min) . BTP2 in2+ responses, we measured Ca2+ oscillations within the ER lumen using a genetically encoded ER-targeted Ca2+ sensor, D1ER [2+ oscillations within the ER lumen of HEK293_RyR2 cells was also positively correlated with increasing [Ca2+]o. Expression of TRIC-A-mCherry significantly reduced the frequency of ER Ca2+ signals by about 50% at each [Ca2+]o as compared to controls (2+ store-depletion events (2+]o). Moreover, TRIC-A clearly increased the time required to obtain complete ER Ca2+ store refilling  inhibitor 2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ), to prevent ER refilling in Ca2+-free external medium to measure Ca2+ release from ER, followed by readdition of 1 mM CaCl2 to the medium to measure Ca2+ entry. In this setting, TRIC-A expression promoted Ca2+ release from the ER  in these cells  to 53% significantly enhanced endogenous SOCE as compared to controls conducted using a scrambled siRNA (si-scr)  . TRIC-A s by 31%  and enha2+ rises . These f2+ signals in wild-type HEK293 cells that lack RyR2 expression (2+]i in response to stimulation with carbachol (CCh) + BHQ  + BHQ  or CCh ah) + BHQ  were not and 35% . Importa and 35% .2+ signaling responses, sustained via SOCE, we investigated [Ca2+]i responses in HEK293 cells at various [CCh]. As reported previously [2+]i responses varied with an increase in stimulus intensities, with baseline Ca2+ oscillations at low [CCh] and sustained elevations at maximal [CCh]. We observed TRIC-A–induced variation in the Ca2+ signaling pattern at each [CCh]. We categorized these responses into 4 groups between 6- and 10-min time frames: no sustained response to CCh or exclusively ER Ca2+ mobilization from stores (no response), oscillations that return to basal levels after each release event (baseline oscillations)  , oscillalations) , and suslations) . When stlations) , 57% of 0 μM CCh , the maj2+]ER during store depletion by caffeine and its subsequent refilling by elevation of [Ca2+]o from nominally free to 1 mM in HEK293_RyR2 cells . Yellow fluorescent protein (YFP)-STIM1 and Orai1-CFP were coexpressed along with mCherry-ER-3 (control) or TRIC-A-mCherry (TRIC-A), and ICRAC was measured using a whole-cell patch-clamp technique  i) was monitored using Fura-2 ratiometric imaging. Briefly, cells on coverslips were loaded with 1 μM Fura-2 AM for 30 min in an experimental buffer, composed of (in mM): 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The coverslip was then mounted in a perfusion chamber on an inverted microscope  with a 20×/0.75 objective and perfused with indicated solutions at 37°C. During the recordings using Live Acquisition v2.6 software , cells were excited alternately using 340/26 and 380/11 nm filters  in an Oligochrome excitation system (FEI), and fluorescent images were captured using 510/84-nm emission filter (Semrock) with an ORCA-03G digital CCD camera . The 340/380 ratio was used as an index of cytosolic Ca2+ levels.Changes in intracellular Ca2+ levels, [Ca2+]ER, were monitored in cells transfected with a CFP/YFP FRET-based D1ER sensor at a 535/470 emission ratio using a 430/24-nm excitation filter, 505dcx dichroic, and 470/24- and 535/30-nm emission filters  with an additional OptoSplit II  in the same setup. Dynamic FRET between STIM1-CFP and YFP-Orai1 was measured using the same setting used for D1ER.ER luminal CaFRET was performed in the same epifluorescence microscope setting as described previously  BSA-TBST) were used: myc , STIM1 , Orai1 , TMEM38A , TMEM38B , and β-actin . After washing, the membranes were incubated with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG  . Immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) in ChemiDoc MP Imaging System (Bio-Rad), and densitometric evaluation was performed using Image Lab 6.0 software (Bio-Rad).Cells were washed with 1×PBS and lysed in ice-cold Pierce IP lysis buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Cell lysates were centrifuged .RNA was isolated from cell lysates using QIAshredder and RNeasy Mini Kit (Qiagen) and reverse transcribed using a high-capacity cDNA reverse transcription kit  in a thermal cycler (Bio-Rad) according to the manufacturer’s protocol. qPCR was performed using QuantiFast SYBR Green RT–PCR kit (Qiagen) in a LightCycler 480 . Relative expression of the target gene was normalized to mouse n represents the number of cells from at least 3 independent experiments unless specified otherwise. The approximate normal distribution of data was assessed by z-value of skewness and kurtosis and D'Agostino–Pearson omnibus normality test. If normally distributed, unpaired t test (2 groups) or one-way ANOVA followed by Dunnett’s multiple comparison test (more than 2 groups) was used to test the statistical significance (with Welch’s correction for significantly different variances between groups); otherwise, Mann–Whitney rank test (2 groups) or Kruskal–Wallis test followed by Dunn’s multiple comparison test (more than 2 groups) was applied. A χ2 test was used to analyze responses in cell populations. All tests were two-tailed, and p values < 0.05 were considered significant.Data analyses were performed using OriginPro 2015  and Prism 5 . Data are expressed as mean ± SEM. S1 Data(XLSX)Click here for additional data file.S1 Fign = 3 independent experiments. TRIC-A-mCherry and β-actin were used as the positive control and loading control, respectively. (B) Representative epifluorescence images of an HEK293 cell expressing mCerulean-ER-5  or CFP-GPI  with TRIC-A-mCherry  along with an overlay (right) of both proteins under basal conditions. Scale bar = 10 μm (C) Traces of [Ca2+]ER-sensitive D1ER FRET ratio, representing SOICR-associated oscillations in an mCherry-ER-3–  or TRIC-A-mCherry–transfected  HEK293_RyR2 cell and lack of oscillations in a 3 μM BTP2-incubated  control cell. (D) Ca2+ oscillation frequency at 0.1, 0.3, and 1 mM [Ca2+]o and (E) amplitude at 1 mM [Ca2+]o in TRIC-A cells (n = 32) versus controls (n = 23); *p < 0.05, ***p < 0.001; bars represent mean ± SEM. Underlying data in panels (C–E) are included in 2+ sensor; ER, endoplasmic reticulum; FRET, Förster resonance energy transfer; GPI, glycosylphosphatidylinositol; HEK293, human embryonic kidney 293; RyR, ryanodine receptor; SOICR, store-overload–induced Ca2+ release; TRIC, trimeric intracellular cation.(A) Representative western blot showing the absence of endogenous TRIC-A bands at approximately 33 kDa using anti-TRIC-A antibody in wild-type (−) and TRIC-A-mCherry–transfected (+) HEK293 cells, (TIF)Click here for additional data file.S2 Fig2+-sensitive Fura-2 ratio represent SOICR-associated oscillations in a (A) control (black) or 2-APB–incubated (red) cell and a (C) YFP (black) or YFP-Orai1-E106Q (red) transfected cell. Bars show mean ± SEM values for Ca2+ oscillation frequency at 0.1, 0.3, and 1 mM [Ca2+]o in (B) 2-APB–incubated cells (n = 98) versus controls (n = 81) and (D) YFP-Orai1-E106Q-transfected cells (n = 43) versus YFP-transfected controls (n = 23), ***p < 0.001. Underlying data in panels (A–D) are included in 2+-sensitive fluorescent indicator; Orai1, Ca2+-release–activated Ca2+ channel 1; RyR, ryanodine receptor; SOICR, store-overload–induced Ca2+ release; YFP, yellow fluorescent protein; 2-APB, 2-Aminoethoxydiphenylborane.Traces of cytosolic Ca(TIF)Click here for additional data file.S3 Fig2+-sensitive Fura-2 traces in mCherry-ER-3  or TRIC-A-mCherry –transfected (A) HEK293_RyR2 cells and (E) RBL-2H3 cells, showing SOCE after ER Ca2+ depletion with 30 μM BHQ. Bar graphs show mean ± SEM values for  SOCE rate,  peak and sustained SOCE amplitude, and (D) ER Ca2+ release peak amplitude in TRIC-A (+) (n = 52) versus control (n = 54) HEK293_RyR2 cells and TRIC-A (+) (n = 25) versus control (n = 27) RBL-2H3 cells; *p < 0.05, **p < 0.01, ***p < 0.001. Underlying data in panels (A–G) are included in t-butyl-1,4-benzohydroquinone; ER, endoplasmic reticulum; Fura-2, cytosolic Ca2+-sensitive fluorescent indicator; HEK293, human embryonic kidney 293; ns, nonsignificant; RBL-2H3, rat basophilic leukemia cell line; RyR, ryanodine receptor; SOCE, store-operated Ca2+ entry; TRIC, trimeric intracellular cation.Average cytosolic Ca(TIF)Click here for additional data file.S4 FigTRIC-A in HL-1 cells transfected with si-scr or si-TRIC-A and normalized to the housekeeping gene GAPDH. n = 3 independent experiments. (B) Traces of cytosolic Ca2+-sensitive Fura-2 ratio in HL-1 cells, transfected with si-scr or si-TRIC-A, showing SOCE after SR Ca2+ depletion with 10 mM caffeine + 30 μM BHQ. Bars show (C) SOCE rate and (D) peak and sustained SOCE amplitude in si-TRIC-A (n = 361) versus si-scr (n = 375) transfected cells, *p < 0.05. Underlying data in panels (A–D) are included in t-butyl-1,4-benzohydroquinone; Fura-2, cytosolic Ca2+-sensitive fluorescent indicator; HL-1, mouse atrial muscle cell line; si-scr, scrambled small interfering RNA; SOCE, store-operated Ca2+ entry; SR, sarcoplasmic reticulum; TRIC, trimeric intracellular cation.(A) Bars show mRNA expression of (TIF)Click here for additional data file.S5 Fig2+-sensitive Fura-2 traces in mCherry-ER3  or TRIC-A-mCherry –transfected HEK293 cells, showing 100 μM CCh-induced Ca2+ depletion from IP3R stores. (B) Bar graphs show peak amplitude of IP3R store-Ca2+ release in TRIC-A (+) (n = 54) versus control (n = 79) HEK293 cells. (C) Average cytosolic Ca2+-sensitive Fura-2 traces in mCherry-ER-3  or TRIC-A-mCherry –transfected HEK293 cells, showing SOCE after ER Ca2+ depletion with 1 μM thapsigargin. Bar graphs show (D) ER Ca2+ release peak amplitude, (E) SOCE rate, and (F) peak and sustained SOCE amplitude in TRIC-A (+) (n = 96) versus control (n = 116) HEK293 cells. *p < 0.05; mean values ± SEM are shown. Underlying data in panels (A–F) are included in 2+-sensitive fluorescent indicator; HEK293, human embryonic kidney 293; IP3R, inositol 1,4,5-triphosphate receptor; ns, nonsignificant; SOCE, store-operated Ca2+ entry; TRIC, trimeric intracellular cation.(A) Average cytosolic Ca(TIF)Click here for additional data file.S6 Fign = 6 independent experiments. (B) Densitometric evaluation of immunoreactive bands of Orai1 and STIM1 shown in (A). (C) Epifluorescence of overexpressed Orai1-CFP and YFP-STIM1 in HEK293 cells coexpressing mCherry-ER-3 (control) or TRIC-A-mCherry (TRIC-A), n = 18 in each group. Bars represent mean ± SEM. Underlying data in panels B and C are included in 2+-release–activated Ca2+ channel 1; STIM1, stromal interaction molecule 1; TRIC, trimeric intracellular cation; YFP, yellow fluorescent protein.(A) Representative western blots for STIM1, Orai1, and TRIC-A-mCherry expression in control and TRIC-A-mCherry–transfected HEK293 cells with β-actin as a loading control, (TIF)Click here for additional data file.S7 Figt-butyl-1,4-benzohydroquinone; CCh, carbachol; Co-IP, coimmunoprecipitation; HEK293, human embryonic kidney 293; STIM1, stromal interaction molecule 1; TRIC, trimeric intracellular cation.Representative Co-IP of endogenous STIM1 with myc-TRIC-A expressed in HEK293 cells. Lysates were obtained from basal (−) and ER-depleted (+) (100 μM CCh + 30 μM BHQ) HEK293 cells, n = 3 independent experiments. BHQ, 2,5-Di-(TIF)Click here for additional data file.S8 Fign = 8 from 3 different experiments. (D) Traces of cytosolic Ca2+-sensitive Fura-2 ratio, represent constitutive Ca2+ influx, leading to sustained cytosolic Ca2+ rise, but not oscillations, in HEK293 cells expressing YFP-STIM1-D76A  and lack of any constitutive Ca2+ influx and oscillations in HEK293 cells ; mean ± SEM values are shown. Underlying data in panels (B–D) are included in t-butyl-1,4-benzohydroquinone; CCh, carbachol; ER, endoplasmic reticulum; Fura-2, cytosolic Ca2+-sensitive fluorescent indicator; HEK293, human embryonic kidney 293; ns, nonsignificant; SOICR, store-overload–induced Ca2+ release; STIM1, stromal interaction molecule 1; TIRF, total internal reflection fluorescence; TRIC, trimeric intracellular cation; YFP, yellow fluorescent protein.(A) Representative TIRF images of a basal (top) and ER-depleted (100 μM CCh + 30 μM BHQ) (bottom) HEK293 cell expressing YFP-STIM1-D76A  and TRIC-A-mCherry  with an overlay (right) of both proteins. Scale bar = 5 μm. (B) Line scans of YFP-STIM1-D76A and TRIC-A-mCherry in a basal and ER-depleted cell shown in (A). (C) Bars show mean ± SEM values of Mander’s coefficient for proportion of TRIC-A-mCherry colocalized with STIM1 under basal and ER-depleted conditions, (TIF)Click here for additional data file.S9 Fign = 18 each). Overall size distribution in TRIC-A cells was significantly different from that in controls . (C) Mean ± SEM of puncta density (number/μm2) of Orai1-CFP and YFP-STIM1 in TRIC-A (+) cells compared to controls. Underlying data in panels (A–C) are included in 2+-release–activated Ca2+ channel 1; STIM1, stromal interaction molecule 1; TRIC, trimeric intracellular cation; YFP, yellow fluorescent protein.Proportion of punctae (%) of various sizes of (A) Orai1-CFP  and (B) YFP-STIM1  in control and TRIC-A (+) cells ((TIF)Click here for additional data file.S1 VideoTime-lapse TIRF image stack showing clustering of YFP-STIM1 (left) and TRIC-A-mCherry (right) coexpressed in HEK293 cell upon ER Ca2+depletion, related to  ER, endoplasmic reticulum; HEK293, human embryonic kidney 293; STIM1, stromal interaction molecule 1, TIRF, total internal reflection fluorescence; TRIC, trimeric intracellular cation; YFP, yellow fluorescent protein.(MP4)Click here for additional data file.S2 VideoER, endoplasmic reticulum, HEK293, human embryonic kidney 293; TIRF, total internal reflection fluorescence; TRIC, trimeric intracellular cation.(MP4)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.