An optical fiber 200 μm in diameter can detect the coherent activity of a subpopulation of neurons. In layer 5 of the M1 and V1, we showed that population Ca2+ transients reliably occurred preceding the impending locomotion. Interestingly, the M1 Ca2+ transients started ~100 ms earlier than that in V1. Furthermore, the population Ca2+ transients were robustly correlated with head movements. Thus, our work provides a simple but efficient approach for monitoring the cortical Ca2+ activity of a local cluster of neurons during locomotion in freely moving animals.Locomotion involves complex neural activity throughout different cortical and subcortical networks. The primary motor cortex (M1) receives a variety of projections from different brain regions and is responsible for executing movements. The primary visual cortex (V1) receives external visual stimuli and plays an important role in guiding locomotion. Understanding how exactly the M1 and the V1 are involved in locomotion requires recording the neural activities in these areas in freely moving animals. Here, we used an optical fiber-based method for the real-time monitoring of neuronal population activities in freely moving mice. We combined the bulk loading of a synthetic Ca We reliably observed Ca2+ signals preceding locomotion in both regions. Interestingly, M1 signals occurred earlier than the V1 signals. Finally, we found that these population Ca2+ signals were highly correlated with head movements.Locomotion is a basic behavior of animals that involves neural activity in many cortical and subcortical networks . All experimental procedures were performed according to institutional animal welfare guidelines and were approved by the Third Military Medical University Animal Care and Use Committee.2+ signal measurements  was excited at 488 nm by a solid-state laser. The light intensity was approximately 0.22 mW/mm2 at the tip of the fiber. The fluorescence emission was detected with an avalanche photodiode . The specialized optic setup was designed to accommodate the simultaneous recording of Ca2+ signals. The laser light on/off control and the data acquisition were managed using self-customized software on the LabVIEW platform .A custom-built fiber setup was used for the neuronal Ca2 and then placed in a stereotactic head frame on a heating pad, where the animals were anesthetized with isoflurane until the surgery ended. The eyes were protected by ophthalmic ointment to prevent drying. A small craniotomy (0.5 × 0.5 mm) was made above the cortical area after removing the hair and skin. The coordinates of the craniotomy were as follows: for the M1 (from bregma): AP 0 mm, ML 1 mm (relative to midline); for the V1: AP 3 mm, ML 2.5 mm. A glass micropipette with a tip diameter of approximately 10 μm was placed directly above the skull and filled with OGB-1AM solution. Approximately 80–100 nl of solution was injected into the tissue at a depth of 600 μm . Following each injection, the pipette was kept in place for an additional 5 min before being slowly withdrawn.The animals were anesthetized with 1.5% isoflurane in pure OApproximately 30 min after dye application, a 200-μm-diameter optical fiber with a numerical aperture of 0.48  was inserted into the stained region with a micromanipulator. The fiber was glued into a short cannula  to maintain stability. To provide maximal fluorescence intensity, the fiber was advanced typically up to 550 μm below the cortical surface. At the beginning, the anesthesia level was adjusted to 2%. After the animals had adapted for 20 min and their respiration rates remained stable, the recording experiments began. After continuous recording for approximately 10 min, anesthesia was adjusted to a lower level. We then recorded again for another 10 min as described above. Recordings were obtained from each mouse under 2–3 anesthesia levels.2+ signals and locomotion were recorded simultaneously while the animals were freely moving. Every mouse was continuously recorded from approximately 40 min to 1 h. The Ca2+ transients were sampled at 2000 Hz with customized acquisition software based on the LabVIEW platform . The videos were recorded at 30 Hz at a spatial resolution of 1280 × 720 pixels . This frame rate may lead to a maximal system error of 33 ms when we calculated the response latency. All the Ca2+ transients and behavior videos were synchronized offline using event marks.After obtaining recordings from mice under anesthesia, the cannula and skull were fixed together using dental cement. After 20 min of solidification, the mice were moved back to their original house. Following recovering for approximately 2 h, the mice were placed into a white rectangular box (29 × 17 cm) in which they could move freely. A camera was placed just above the box and recorded the movements of the mice. Neuronal CaTo document the OGB-1 staining and confirm the relative position of the optical fiber, all recorded mice were perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS) after the experiments. Brain samples were dehydrated with 15% sucrose in PBS for 24 h. Then, the brain samples were sectioned into 30-μm-thick slices, and 4′,6-diamidino-2-phenylindole was used to stain nuclei. Images were acquired using a fluorescence microscope and a 4× objective with a numerical aperture of 0.13.2+ transients were acquired at a sampling rate of 2000 Hz after being converting into electrical signals through the Si APD. The data were low-pass filtered with a Savitzky-Golay finite-impulse response smoothing filter with 50 side points and a polynomial order of 3. Then, ΔF/F = (f − fbaseline)/fbaseline, relative fluorescence changes, were calculated as Ca2+ transients, where the fbaseline was the baseline level of fluorescence determined during the current recording period of the test. Ca2+ transients were automatically detected with a template-matching algorithm, taking into account the properties of rise and decay times of the Ca2+ signals. A Ca2+ transient was accepted as a signal when its amplitude was greater than three times the standard deviation of the noise band.CaThe mouse movement was calculated by the change in video clip image relative to its body size. Image frames from the video clip were converted into binary format to get the mouse shape according to its image intensities. A logical “OR” operation was performed between two consecutive frames to get the pixel size of absolute change of the mice. The ratio of such image change relative to the mouse body size, which was calculated beforehand, was defined as the mouse movement. This scoring procedure cannot differentiate the head movement from the general movement. Therefore, the head movements in the last figure were identified by eyes.p < 0.05 was considered significant.Statistical analysis was conducted in MATLAB . For all statistical tests, significance was measured against an alpha of 0.05. The level of 2+ activity in cortical neurons stained with the synthetic Ca2+ indicator OGB-1AM. We used a previously described fiber recording device . The quantitative analysis indicated that the population Ca2+ activity was strongly dependent on the level of anesthesia. In M1, the frequency increased from 0.04 ± 0.01 Hz to 0.45 ± 0.03 Hz with a decrease in the isoflurane concentration . In addition, the recordings of population activity in V1 revealed that no difference was found between M1 and V1 in frequency, amplitude and rise time of the Ca2+ transients at the same anesthesia level .Approximately 10 min after dye injection, we implanted an optical fiber with a diameter of 200 μm above the stained cortical area Figure . Similarn Figure . Howevern Figure . In cont2+ transients and the body movements, we recorded Ca2+ activities in layer 5 neurons of the M1 in freely behaving mice in a white, opaque, rectangular chamber  and resting  states. In this example, when the mouse was moving freely, Ca2+ transients were observed with a high frequency, while almost no transients were observed in the resting states. On average, both the frequency  and amplitude  of the Ca2+ transients in the moving states were significantly higher than those in resting states. As a control, these signals were not seen in the mice whose layer 5 neurons expressed green fluorescent protein . In addition, when we compared the amplitude of the population Ca2+ transients in the M1 and V1, there was no significant difference . Furthermore, we found that, compared to that under light conditions, in the dark both the amplitude and the correlation of movements and Ca2+ signals significantly decreased  or the V1 . In contrast, almost no Ca2+ transients were observed during resting (quiescent) states . During locomotion, we found that Ca2+ transients reliably occurred approximately 100 ms before the onset of movement and then persisted throughout the whole process of each movement. This activity was similar to a previously defined activity in the monkey motor cortex, referred to as perimovement activity (Churchland et al., In this study, we applied an optical fiber-based Ca2+ transients preceding impending movement also occurred in the layer 5 neurons of the V1. Interestingly, such activity in the V1 started ~100 ms later than that in M1. Previous studies have reported this preceding activity in upper layers as well as in deep layers using electrophysiological recordings in the V1 (Ayaz et al., Similar to the results in the M1, we found that population Ca2+ dyes, we could achieve a recording of activity in a highly restricted area approximately 300–500 μm in diameter. In many other studies (Lütcke et al., 2+ or voltage sensors allowed the activity of cell type-specific neural populations to be recorded during free behavior. Using this simple but efficient approach, we studied locomotion-related population signals of neurons in deep layers of both the M1 and V1. In the near future, the application of endoscope-based imaging techniques with cellular resolution (Jung et al., Population activity plays an important role in many brain functions, such as information processing, learning, memory and behavior. For example, extensive studies have focused on slow oscillations, i.e., slow population activity at a frequency less than 1 Hz mainly occurring during sleep and anesthesia (Steriade et al., 2+ activity in freely behaving animals. Using this method, we provide insights into body movement-related population neuronal activity in both the M1 and V1, which could be the first step toward understanding cortical information processing during locomotion.In summary, our results illustrate that the optical fiber-based approach is an efficient method for monitoring cortical CaQZ, JYao, YG, HJ, JYan, ZF, WL and XC contributed to the design of the study and interpretation of the data. QZ, JYao, YG, HQ, JZ, JP and HJ performed the experiments and acquired the data. QZ, SL, JG, XL, WJ and HQ processed and analyzed the data. QZ, ZF, WL and XC wrote the manuscript with help from all the other authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mutating a stretch of C-terminal Ca2+-binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca2+] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca2+-dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression.MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca MicroRNAs (miRNAs)2+ influx and trigger relevant signaling cascades  subunits –10. For –2+-sensing protein that responds to NMDAR-mediated Ca2+ influx to regulate its interaction with the AMPAR subunit GluA2. The increased Ca2+ binding to PICK1 upon NMDAR activation results in an enhanced interaction between PICK1 and GluA2, which facilitates AMPAR internalization  and PDZ domain protein that regulates the trafficking of AMPARs in LTD and LTP –13. Furt–lization , 15. Prelization . Because2+-sensing property of PICK1 is required for regulating its interaction with Ago2. We show that, in high [Ca2+]free, PICK1-Ago2 complexes are disrupted. Furthermore, we show that, when the PICK1 Ca2+-binding site is mutated, the cLTD-induced increase in the activity of dendritically localized miRNAs in neurons is blocked. This study describes a novel mechanism for the regulation of Ago2 activity by Ca2+ in response to NMDAR activation in neurons, which we propose might be involved in initiating changes in protein expression required for LTD.In this study, we demonstrate that the Ca2+ sensor that interacts with the AMPAR subunit GluA2 in a Ca2+-dependent manner . We chose these [Ca2+]free because PICK1 shows Ca2+ regulated binding properties over this range of [Ca2+]free for GluA2 .PICK1 has been identified previously as a Cat manner . Becauset manner , we invend 16 μm A. We choor GluA2 . Indeed,eviously . In contto 16 μm A.2+]free. PICK1 immune complexes from DIV14 cortical neuronal extracts were isolated under varying [Ca2+]free (B). As with our His6 pulldown experiments, we observed a significant increase in Ago2 binding to PICK1 when the [Ca2+]free was increased from 0 to 4 μm but a significant decrease at 16 μm [Ca2+]free. Consistent with previous findings, more GluA2 bound to PICK1 isolated under higher [Ca2+]free  in the PDZ domain that disrupt GluA2 binding (A) (B).Because Ago2 and GluA2 bind PICK1 with approximately opposing [Ca binding A 17). I. I2+] deding (A) . In contding (A) B.2+  renders the N-terminal site insensitive to Ca2+  to prevent the C-terminal region from binding Ca2+ (A).PICK1 contains two acidic regions that have been demonstrated previously to bind Ca2+ , 15. One2+ . To idening Ca2+ A.B), suggesting that the C-terminal acidic region of PICK1 regulates Ago2 binding. To confirm that the C-terminal acidic region of PICK1 regulates Ago2 binding, we performed co-IP experiments in HEK293 cells co-transfected with Myc-Ago2 and FLAG-PICK1 Δ380–390, in which residues 380–390 were deleted (C). FLAG-PICK1 Δ380–390 showed a similar interaction with Myc-Ago2 as FLAG-PICK1 A×10 (D).When FLAG-tagged versions of these mutants were co-transfected into HEK293 cells with Myc-Ago2, co-immunoprecipitations showed that FLAG-PICK1 D3N bound Myc-Ago2 at similar levels compared with FLAG-PICK1 WT. Interestingly, the interaction of Ago2 with FLAG-PICK1 A×10 was significantly greater compared with FLAG-PICK1 WT and D3N B, sugges deleted C. FLAG-PCK1 A×10 D.2+-dependent Ago2 binding, we performed GST pulldown experiments on cortical neuronal extracts in defined [Ca2+]free using the C-terminal tail of PICK1 (GST-Δ354) as bait (A). Ago2 binding to GST-Δ354 displayed a biphasic dependence on [Ca2+]free, consistent with our pulldown and co-IP experiments shown in m [Ca2+]free and the weakest at 16 μm [Ca2+]free. Interestingly, Ago2 binding to GST-Δ354 A×10 did not respond to changes in [Ca2+]free, indicating that the Ca2+−sensitive Ago2-PICK1 interaction requires amino acids 380–390 of PICK1 (B). Together, these experiments demonstrate that the PICK1-Ago2 interaction is regulated by Ca2+ binding to the C-terminal acidic region of PICK1 located within the Ago2 binding domain.To investigate the role of PICK1 residues 380–390 in Ca as bait A. Ago2 bof PICK1 B. Togeth2+ entry through NMDARs increases localized intracellular [Ca2+]. Therefore, to determine whether the Ca2+-sensing property of PICK1 is required for mediating the disassociation of Ago2 during cLTD, we performed molecular replacement experiments in cortical neurons, where endogenous PICK1 was knocked down with a previously characterized shRNA for PICK1 and replaced with sh-resistant GFP-PICK1 WT or GFP-PICK1 A×10 . To investigate whether these constructs altered the neuronal architecture, we performed a Sholl analysis to measure dendritic branching. PICK1 knockdown decreased the number of dendritic intersections 70–100 μm away from the cell body in a similar manner to a previous report . Furthermore, neither PICK1 knockdown nor molecular replacement altered total dendritic length . Together, these experiments suggest that the C-terminal Ca2+-binding properties of PICK1 do not regulate dendritic branching or dendritic length.Previously, we demonstrated that the PICK1-Ago2 interaction is significantly reduced after the induction of cLTD . During CK1 A×10 , 16. shPs report . This phA and B). 10 min after cLTD induction, Ago2 binding to PICK1 WT was significantly reduced, as seen previously (A and B). In contrast, cLTD caused a similar increase in GluA2 interaction with both PICK1 WT and the A×10 mutant (A and C). These data indicate that the Ca2+-sensing property of the PICK1 C-terminal acidic region underlies the release of Ago2 from PICK1 following cLTD induction but that it is not involved in regulating the interaction with AMPARs.We performed GFP-trap experiments to measure the interaction between GFP-PICK1 and endogenous Ago2 under basal and cLTD conditions. Under basal conditions, similar levels of Ago2 bound to PICK1 WT and A×10 , A and Beviously . Howevereviously , A and B0 mutant , A and C2+ sensing by PICK1 triggers Ago2 release from PICK1-positive endosomes. To test this, we examined Ago2 co-localization with GFP-PICK1 WT or A×10 under basal or cLTD conditions. Although we did not detect any difference in the degree of co-localization of Ago2 with GFP-PICK1 WT or A×10 under basal conditions, cLTD caused a significant reduction in co-localization between GFP-PICK1 WT and Ago2, which was completely blocked by the A×10 mutation (A and B). Together, these experiments demonstrate that the C-terminal acidic region of PICK1 is essential for regulating the disassociation of PICK1 from Ago2 in response to cLTD.PICK1 localizes to endosomal compartments , and we mutation , A and B2+, we examined endogenous PICK1-Ago2 binding in cortical neurons stimulated with NMDA in the absence of extracellular Ca2+. NMDAR activation significantly reduced PICK1-Ago2 binding by ∼50% in the presence of normal (1.8 mm) Ca2+. In contrast, NMDAR stimulation in the absence of extracellular Ca2+ had no effect on PICK1-Ago2 binding, indicating that extracellular Ca2+ is the primary source of Ca2+ that disrupts PICK1-Ago2 interactions during cLTD (A). We also confirmed that our stimulation protocol induced cLTD by analyzing GluA1 phosphorylation at serine 845. Dephosphorylation at this site by the Ca2+-dependent phosphatase calcineurin is a hallmark of LTD (2+ but was unchanged in the absence of extracellular Ca2+ (B). This demonstrated successful induction of cLTD in the presence of extracellular Ca2+.To investigate whether the dissociation of Ago2 from PICK1 caused by cLTD induction required extracellular Caing cLTD A. We alsk of LTD , 22. Glular Ca2+ B. This dA). Serine 845 dephosphorylation followed a similar time course (B). Together, these experiments suggest that extracellular Ca2+ entry through NMDARs disrupts PICK1-Ago2 complexes very rapidly after stimulation.To further define the dissociation of Ago2 from PICK1 in response to cLTD induction, we performed a time course experiment to analyze endogenous PICK1-Ago2 complexes at 0, 5, 10, and 30 min after NMDAR stimulation. Interestingly, a significant reduction in PICK1-Ago2 binding was observed immediately after stimulation with no further reduction being observed at the later time points A. Serinee course B. Togeth2+ binding region of PICK1 is involved in the cLTD-induced increase in Ago2 function, we measured the activity of a number of miRNAs using luciferase reporter assays. The 3′ UTRs of LIMK1, APT1, and LIN41 contain miRNA binding sites for miR134, miR138, and Let7, respectively , indicating that the A×10 mutation has no effect on miRNA activity under basal conditions. In response to cLTD induction, we observed a ∼50% reduction in luciferase activity 10 min after stimulation, as reported previously . Luciferase activity was not significantly affected under basal or cLTD conditions with any PICK1 knockdown or replacement background when the miR134- and miR138-binding sites were mutated in the LIMK1 and APT1 reporter constructs so that they could no longer bind miRNA (B and D). PICK1 knockdown or molecular replacement with GFP-PICK1 A10 did not alter Let7 activity, as measured using the LIN41 luciferase reporter under basal or cLTD conditions (E and F). Taken together, these experiments suggest that the C-terminal Ca2+-binding region of PICK1 regulates dendritic miRNA-mediated gene silencing events in response to cLTD induction.We reported previously that cLTD enhances miRNA-mediated gene silencing of the dendritically localized miR134 in a PICK1-dependent manner , suggestectively –25. Whenctively ––26. PICKCK1 A×10 A, indicaeviously . Gene sir manner C. Lucifend miRNA , B and Dnditions , E and F2+ ions. We demonstrate that amino acids 380–390 in PICK1 are responsible for regulating the Ago2-PICK1 interaction in a Ca2+ dependent manner and that mutating this C-terminal Ca2+-binding region of PICK1 blocks miR134- and miR138-dependent translational repression in response to cLTD induction.Here we have identified a novel molecular mechanism that regulates the interaction between PICK1 and the miRISC protein Ago2. We showed previously that PICK1 inhibits Ago2 function in neuronal dendrites and that, during cLTD, Ago2 is released from PICK1, causing an increase in miRNA-mediated translational repression . In this2+]free of 16 μm compared with zero Ca2+. However, the interaction is strongest at 4 μm [Ca2+]free, suggesting a biphasic dependence on [Ca2+]. The PICK1-GluA2 interaction also has a biphasic dependence on [Ca2+], but, in contrast to PICK1-Ago2, binding increases between 0 and 15 μm, with decreased PICK1-GluA2 interaction again at 30 μm [Ca2+]free  10–13 (unless otherwise stated) using Lipofectamine 2000 (Invitrogen) and used for experiments 2–5 days post-transfection. Chemical LTD was induced by bath application of 50 μm NMDA plus 20 μm glycine in HBS ) for 3 min at 37 °C and harvested or fixed 10, 30, or 60 min later for biochemistry, luciferase assays, or imaging. Where cLTD was induced in the absence of Ca2+, cortical cultures were washed twice with HBS containing zero CaCl2 before bath application of 50 μm NMDA in the same buffer.HEK293 cells were cultured in complete DMEM (Gibco) and passaged at ∼70–80% confluency. HEK cells were transfected with Jetpei (PolyPlus) transfection reagent according to the instructions of the manufacturer. Rat embryonic cortical neuronal cultures were prepared from embryonic day 18 Wistar rats using standard procedures. The culture medium was Neurobasal medium (Gibco) supplemented with B27 (Gibco) and 2 mCells grown on coverslips were fixed in 4% paraformaldehyde (Thermo Fisher) in PBS (Sigma) supplemented with 2% sucrose at room temperature. Cells were permeabilized in 0.5% Nonidet P-40 for 2 min. Coverslips were blocked in 3% BSA (Sigma) for 1 h and incubated with anti-PICK1  in 3% BSA for 1 h at room temperature, followed by goat anti-rabbit Alexa Fluor 568 for 45 min  and mounted in DAPI-free medium (Sigma) onto slides. Coverslips were imaged on a Leica SP5 confocal system under a ×63/1.4 numerical aperture (NA) oil immersion or ×40/1.25 numerical aperture (NA) oil immersion objective using filters set up to image GFP, mRUBY, or Alexa Fluor 568. The Leica application suite software was used to acquire 0.37-μm stepped Z stacks throughout the depth of the cells, and maximal intensity projections generated by ImageJ Fiji are presented in this work. Image processing and co-localization analyses were performed using ImageJ and Coloc2. Three randomly selected dendrites were analyzed per neuron. Mander's fractions were measured using thresholding, values were normalized for each of at least three independent experiments, and statistical significance was determined using two-way ANOVA. All the error bars on graphs represent standard deviation of the mean. Sholl analysis was performed by thresholding the mRUBY channels and using the Sholl plugin (ImageJ Fiji) to measure the number of dendritic intersections every 10 μm over a total radius of 150 μm from the cell body. The numbers of intersections were measured from three independent neuronal cultures. Total dendritic length was measured using the Simple Neurite Tracer plugin (ImageJ Fiji) to calculate the total dendrite length of a neuron based on filling neurons with mRUBY. Traces were taken from three independent experiments, and statistical significance was determined using one-way ANOVA.Levels of PICK1 knockdown and molecular replacement were quantified by measuring integrated densities of PICK1 clusters detected using anti-PICK1 in cortical neuronal dendrites using ImageJ (Fiji).m NaCl, and 20 mm HEPES (pH 7.4)), and 1% of extract was removed for input. The remaining lysate was precleared with protein G-Sepharose beads  at 4 °C for 1 h. 400 μg of cell lysates was incubated with 2 μg of anti-PICK1 (NeuroMab clone L20/8), anti-FLAG , or control IgG  antibodies and pulled down with protein G-Sepharose beads  at 4 °C for 1 h. Beads were washed three times (1 min each) in 1 ml of IP buffer at 4 °C. Bound proteins were detected by Western blotting. For co-IPs carried out under defined [Ca2+]free, 8–10 6-cm dishes of cortical neuronal cultures were pooled together in 1 ml of co-IP buffer with zero Ca2+. 1% of this pool was taken as input, and the remaining extract was equally divided and diluted into 5 ml of co-IP buffer with the appropriate [Ca2+]total and [EGTA] to give the stated [Ca2+]free. Therefore, these experiments share the same input. Defined [Ca2+]free were determined using the Max chelator software (Stanford).Cells were lysed in IP buffer . Bacteria were sonicated on ice, and debris was removed by centrifugation. The resulting supernatant was incubated with glutathione-Sepharose 4B beads  or nickel-nitrilotriacetic acid beads (Qiagen) for 1 h at 4 °C. The beads were washed with 1 ml of HTG buffer three times (1 min each). 8–10 6-cm dishes of cortical neuronal cultures or four T75 flasks of transfected HEK293 cells were pooled together in 1 ml of co-IP buffer with zero Ca2+. 1% of this pool was taken as input, and the remaining extract was equally divided and diluted into 5 ml of co-IP buffer with the appropriate [Ca2+]total and [EGTA] to give the stated [Ca2+]free. This extract was then incubated with recombinant protein immobilized on beads for 1 h at 4 °C. This was followed by washing the beads three times (1 min each) in 1 ml of the appropriate [Ca2+]free co-IP buffers. Bound proteins were detected by Western blotting.Recombinant proteins were expressed and purified from BL21 bacterial cultures. 1 liter of bacterial culture was induced for 2 h using 0.2 mm Tris (pH 7.5), 150 mm NaCl, 0.5 mm EDTA, and 0.5% Nonidet P-40) on ice. 1% was taken as input, and the remaining extract was incubated with 50 μl of GFP-trap beads for 1 h at 4 °C. The beads were then washed with 500 μl of wash buffer (10 mm Tris (pH 7.5), 150 mm NaCl, and 0.5 mm EDTA) three times (1 min each) at 4 °C, and bound proteins were detected by Western blotting.For GFP-trap (Chromotek) pulldowns, six wells of a 6-well plate of DIV10 cortical neurons were transfected with pXLG3-GFP PICK1 constructs and harvested 5 days post-transfection. GFP-trap was performed according to the instructions of the manufacturer. Briefly, cells were lysed in 500 μl of lysis buffer , and bands were visualized using ECL Western blotting substrates . Where appropriate, the membrane was stripped with Restore Western Blot Stripping Buffer (Thermo Fisher) and reprobed. Membranes were incubated with the following primary antibodies overnight at 4 °C: anti-Ago2 , anti-GluA2 , anti-PICK1 , anti-FLAG , anti-c-Myc , anti-GAPDH , anti-GST , anti-GFP , anti-GluA1 , and anti-GluA1 Ser(P)-845 . Secondary antibodies conjugated to HRP were from GE Healthcare and used at 1:10,000 dilutions for 45 min at room temperature. For densitometry, Western blot films were scanned and analyzed in ImageJ, followed by the appropriate statistical analysis. All error bars on graphs represent standard deviation of the mean. For pulldowns and co-IPs, bound proteins were normalized to their respective inputs. For GFP-trap experiments, bound proteins were normalized to the amount of GFP-PICK1 detected by anti-GFP in the pulldown.DIV12 cortical cultures were co-transfected with the appropriate luciferase constructs and pXLG3-GFP vectors and prepared for luciferase recordings 48 h post-transfection. The Dual-Luciferase reporter assay system (Promega) was used to perform the assays according to the instructions of the manufacturer. Values were normalized for each of at least three independent experiments, and the appropriate statistical analysis was performed. All error bars on graphs represent standard deviation of the mean.D. R. and J. G. H. designed the study and prepared the manuscript. D. R. performed the experiments. M. F. and G. T. P. prepared recombinant proteins. D. R. analyzed the data.


INaCa) in the t-tubular and surface membranes (INaCa-ratio) computed from the values of INaCa and membrane capacitances (Cm) measured in adult rat ventricular cardiomyocytes before and after detubulation ranges between 1.7 and 25 . Variations of action potential waveform and of calcium turnover within this span of the INaCa-ratio were simulated employing previously developed model of rat ventricular cell incorporating separate description of ion transport systems in the t-tubular and surface membranes. The increase of INaCa-ratio from 1.7 to 25 caused a prolongation of APD  by 12, 9, and 6% and an increase of peak intracellular Ca2+ transient by 45, 19, and 6% at 0.1, 1, and 5 Hz, respectively. The prolonged APD resulted from the increase of INaCa due to the exposure of a larger fraction of Na-Ca exchangers to higher Ca2+ transients under the t-tubular membrane. The accompanying rise of Ca2+ transient was a consequence of a higher Ca2+ load in sarcoplasmic reticulum induced by the increased Ca2+ cycling between the surface and t-tubular membranes. However, the reason for large differences in the INaCa-ratio assessed from measurements in adult rat cardiomyocytes remains to be explained.The ratio of densities of Na-Ca exchanger current ( This resulted in a higher Ca2+ load in SR. However, [Ca2+]st was only slightly affected, because the increase of Ca2+ extrusion from this space, induced by higher INaCa,t, was compensated by increased Ca2+ flux from the cytosol to the t-tubular subsarcolemmal space. Thus, the model showed that a redistribution of Na-Ca exchangers would lead to marked changes in net Ca2+ flux from the surface membrane to the t-tubular membrane through both subsarcolemmal spaces and cytosol .fNaCa,t on cellular electrophysiological activity, we used the model to simulate action potentials, membrane currents, and dynamic changes of Ca2+ concentration in individual intracellular compartments at fNaCa,t set to 0.78, 0.48, and 0.93 at stimulation frequencies of 0.1, 1, and 5 Hz. The results obtained at 0.1 and 1 Hz  and 5 Hz (stimulation frequency corresponding to resting heart rate in rats) are illustrated in Figures To further explore the impact of changing fNaCa,t from 0.78 to 0.48 led to a shortening of APD90 (duration of AP at 90% repolarisation) by approximately 7, 4, and 4% at 0.1, 1, and 5 Hz whereas increasing fNaCa,t to 0.93 resulted in a prolongation of APD90 by 5, 4, and 2%, respectively. However, marked changes were evident in the level of [Ca2+]NSR and in the peak value of transient changes of [Ca2+]c. A decrease of fNaCa,t to 0.48 led to a reduction of [Ca2+]NSR at the end of the cycle by 13, 7, and 3% and to a decrease of peak [Ca2+]c by 15, 8, and 4% at 0.1, 1, and 5 Hz, respectively. Conversely, an increase of fNaCa,t to 0.93 caused a rise of [Ca2+]NSR by 14, 7, and 2% at the end of the cycle and an increase of peak [Ca2+]c by 23, 9, and 2%, respectively. To clarify the mechanisms underlying the described changes induced by redistribution of Na-Ca exchangers, the amounts of Ca2+ moving across sarcolemma, through cytosol, and across SR membrane within a steady-state cycle at 1 Hz stimulation are shown in A redistribution of Na-Ca exchange proteins caused rather smaller but still apparent frequency-dependent change of action potential (AP). A decrease of 2+]NSR that decrease with the increase of stimulation rate (reflecting a decrease of relative changes of SR Ca2+ uptake at shorter stimulation cycle). This resulted in a reduction of the effect on [Ca2+]c at higher stimulation frequencies; the increase of fNaCa,t from 0.48 to 0.93 caused an increase of peak [Ca2+]c by 45, 19, and 6% at 0.1, 1, and 5 Hz, respectively. Hence, the model indicates that the changes in SR Ca2+ load, cytosolic Ca2+ transient, and AP configuration caused by redistribution of Na-Ca exchangers between the t-tubular and surface membranes are potentially important in the whole range of the simulated frequencies explored.These data show that a redistribution of Na-Ca exchangers causes relative changes in ss  and an equivalent increase of Ca2+ release from junctional SR (nrel). Note, however, that because of higher fraction of junctional SR at the t-tubules  with fNaCa,t was not fully compensated by the decrease of Ca2+ diffusion from the t-tubular subsarcolemmal space into the cytosol . Thus, the model shows that the relocation of Na-Ca exchangers from the surface to the t-tubular membrane changes the intracellular gradients of Ca2+ concentration (mainly because of the rise of [Ca2+]ss), which results in an increase of Ca2+ entering the cytosol and SR.The increased  Figures . To explles (0.8 ), the infNaCa,t affected only minutely the total Ca2+ transfers through individual Ca transporters in the model . This implicated that the total transsarcolemmal Ca2+ transfer was nearly unaffected by higher fNaCa,t. Nevertheless, due to the increased Ca2+ cycling, an increased fraction of Na-Ca exchangers at the t-tubules may have a sizeable positive inotropic effect and may play an important role in the adaptation of the heart to the increase in hemodynamic demand during development. Besides, the inverse process could, at least partly, explain the relation between the reduction of cytosolic Ca2+ transient observed at 1 Hz stimulation in cells from failing hearts [INaCa-ratio in the same cells [2+ uptake at shorter stimulation cycle; when fNaCa,t was increased from 0.48 to 0.93, the amount of Ca2+ entering SR during a stimulation cycle increased by 69 and 28% at 0.1 and 1 Hz (see the increase in nup in It is worth mentioning that a change of g hearts  and a deme cells . The freINaCa-ratio affect more the intracellular Ca2+ turnover and related excitation-contraction coupling than the electrical activity of rat ventricular cell in our model. However, the reason of sizeable differences in the INaCa-ratio as evaluated in cardiomyocytes from adult healthy rats remains to be explained.To sum up, changes in the INaCa-ratio in rat ventricular cardiomyocytes deserves attention. The basic characteristics of rats used by Gadeberg et al. [INaCa-ratio of 3.7 in our rat model, which is slightly smaller than the average ratio of 4.3 computed from data by Despa et al. [INaCa-ratio in adult rat ventricular cardiomyocytes is likely affected by other factors. Besides the age of animals, a potentially important role may also be assigned to different experimental conditions and precision in measurement/evaluation of experimental data. The need to explain this physiologically important point strongly calls for new electrophysiological and immunolabelling data from adult rats of different ages.The question about the factors affecting the g et al. , Thomas g et al. , Despa eg et al. , and Yang et al.  are summg et al.  were morg et al.  and Cheng et al.  clearly g et al. . This dia et al.  obtaineda et al. , althouga et al. , exhibitINaCa-ratio in adult rat ventricular cells, as assessed from electrophysiological data published to date, yields strikingly different values. The simulations performed on a model of rat cardiomyocyte showed that such differences in the INaCa-ratio would significantly affect the intracellular gradients of Ca2+ concentration, SR Ca2+ load, and thus the cellular inotropic state. The reason for these differences is however unclear. More studies focused on location and precise assessment of the fraction of Na-Ca exchangers within t-tubules would be of considerable value and would help to explain the differences resulting from available experimental data.The


AML) is a malignant disorder of the myeloid blood lineage characterized by impaired differentiation and increased proliferation of hematopoietic precursor cells. Recent technological advances have led to an improved understanding of AML biology but also uncovered the enormous cytogenetic and molecular heterogeneity of the disease. Despite this heterogeneity, AML is mostly managed by a ‘one‐size‐fits‐all’ approach consisting of intensive, highly toxic induction and consolidation chemotherapy. These treatment protocols have remained largely unchanged for the past several decades and only lead to a cure in approximately 30–35% of cases. The advent of targeted therapies in chronic myeloid leukaemia and other malignancies has sparked hope to improve patient outcome in AML. However, the implementation of targeted agents in AML therapy has been unexpectedly cumbersome and remains a difficult task due to a variety of disease‐ and patient‐specific factors. In this review, we describe current standard and investigational therapeutic strategies with a focus on targeted agents and highlight potential tools that might facilitate the development of targeted therapies for this fatal disease. The classes of agents described in this review include constitutively activated signalling pathway inhibitors, surface receptor targets, epigenetic modifiers, drugs targeting the interaction of the hematopoietic progenitor cell with the stroma and drugs that target the apoptotic machinery. The clinical context and outcome with these agents will be examined to gain insight about their optimal utilization.Acute myeloid leukaemia ( Personalized cancer therapy offers the hope to establish novel and more effective therapeutic standards for patients afflicted with this condition. While traditional chemotherapeutic protocols aim to destroy rapidly dividing cells, but also affect normal  cells, personalized medicine represents a promising concept by which patients whose cancer cells harbour pathophysiologically and therapeutically relevant molecular alterations could be treated with a biomarker‐based ‘targeted’ therapy. In the long term, this strategy may be cost‐effective, even including the required diagnostic and follow‐up tests that accompany therapy . Personalized medicine has become a synonym for the medicine of the future to which many experts ascribe a paradigm change. The overwhelming success of the tyrosine kinase inhibitor imatinib Current standard treatments for AML consist of induction chemotherapy followed by several courses of consolidation chemotherapy or allogeneic stem cell transplantation (aSCT). Herein, induction protocols mostly employ the so‐called 7 + 3 regimen, which entails continuous infusion cytarabine given over 7 days and 3 days of an anthracycline, typically either daunorubicin or idarubicin. Although the ideal dose of daunorubicin remains an open question, this approach has remained unchanged for the past several decades via accelerated approval in 2000 for older patients with AML in first relapse P‐glycoprotein expression, with favourable response rates of 35–44%. Unfortunately, a randomized study where GO was added to low‐dose cytarabine did not translate into improved survival Gemtuzumab ozogamicin (GO), an anti‐CD33 immunoconjugate, has the unique distinction of being the first targeted agent in AML that was approved by the FDA mut patients approached those historically seen in KITwt patients, suggesting that dasatinib may overcome the negative prognostic effect of the KIT mutation. The AMLSG group is conducting a randomized phase III study adding dasatinib to induction chemotherapy in CBF AML. A French Intergroup study showed dasatinib used as single‐agent maintenance failed to prevent relapse in patients with poor molecular response or molecular recurrence following chemotherapy Approximately 25% of core biding factor (CBF) AML patients carry gain‐of‐function mutations in the KIT gene. These mutations result in a constitutively active tyrosine kinase that contributes to aggressive leukaemia growth, and is associated with unfavourable outcome Midostaurin is a multikinase inhibitor that claims the unique distinction of being the first FLT3 inhibitor proven to improve overall survival (OS) in FLT3/ITD‐mutated AML. As a single agent, Midostaurin treatment of 95 patients resulted in 1 partial and no complete remissions versus 26 months for the placebo group (P = 0.007). In addition, a 23% reduction in the risk of death was observed. The landmark results of this trial resulted in its FDA approval in combination with chemotherapy in AML patients younger than 60 years of age in April 2017. It is interesting to note that response rates to induction therapy did not differ significantly between treatment arms, suggesting prolonged exposure is required to benefit from the inhibitor. Moreover, patients randomized to midostaurin who underwent aSCT during the first remission had a survival curve plateau in the 60–70% range suggesting that aSCT remains a very relevant consideration in this population. Another interesting compound, sorafenib, was originally developed as an inhibitor of the serine/threonine kinase Raf but leukaemia clinical trials and physicians have capitalized on its off‐target inhibition of FLT3. Being FDA approved for hepatocellular carcinoma, it is the most widely accessible FLT3 inhibitor in clinical practice and frequently used off‐label. In younger patients, the addition of sorafenib to chemotherapy was well tolerated and showed preferential activity in FLT3‐mutated patients Quizartinib, an exquisitely specific FLT3 inhibitor, has a significantly longer half‐life than the above agents, as well as a greater capacity for inhibition of mutated FLT3 Crenolanib, a drug originally developed as an inhibitor of platelet‐derived growth factor receptor, has shown both activities in FLT3/ITD‐mutated AML and FLT3/ITD D835‐mutated AML Gilteritinib, an agent with activity against wild‐type FLT3, FLT3/ITD, FLT‐TKD D835 and F691, as well as Axl, has been examined for the treatment of relapsed/refractory AML in two early‐phase clinical trials. In the phase I/II CHRYSALIS dose escalation trial, gilteritinib produced an overall response rate of 57% in FLT3‐mutated patients and 63% in patients with FLT3 mutations who received a dose of 80 mg per day or greater et al. −2, 10−3 (major molecular response), or were MRD negative demonstrated a significantly longer median overall survival compared to patients who did not achieve a molecular response, suggesting that the ITD signal ratio may serve as a predictor of durable clinical benefit of gilteritinb.The negative prognostic impact of the fms‐like tyrosine kinase receptor‐3 internal tandem duplication mutation (FLT3/ITD) on AML outcome and its physiologic effect of constitutive signalling through a receptor tyrosine kinase make it a highly desirable drug target. Mutational burden appears to predict addiction to FLT3 signalling and thus response to FLT3 inhibition RAS as well mutations and/or overexpression of upstream receptor tyrosine kinases such as FLT3. RAS inhibitors have had an underwhelming impact on AML. A phase 3 trial evaluating the farnesyltransferase inhibitor tipifarnib as first‐line therapy in older patients resulted in a CR rate of only 8%, and no survival benefit. A phase 2 trial of single‐agent selumetinib In AML, the RAS pathway is activated both by mutations occurring in versus 13.3%) in a randomized phase 2 study Polo‐like kinases (Plks) are involved in mitotic checkpoint regulation and cell division Alvocidib, a potent inhibitor of serine‐threonine Cyclin‐dependent kinases (CDKs) 9, 4 and 7, has been shown to be an active agent against AML Dysregulation of apoptosis in AML is partly mediated by overexpression of the anti‐apoptotic protein BCL‐2 and related family members. Venetoclax (ABT‐199) is a ‘BH3‐mimetic’ antagonist of BCL‐2. A phase 2 study of 32 patients with relapsed/refractory AML reported 5 CRs, the majority of which occured in patients carrying IDH1 or IDH2 mutations. The responses, however, were short‐lived via CXCR4/CXCL12 signalling. In a phase 2 study, 46 patients treated with plerixafor in combination with chemotherapy showed a response rate of 46% (CR+CRi) associated with twofold mobilization in leukaemic blasts into the peripheral circulation Most of the progress in targeting AML–stroma interactions has been made by the development of CXCR4 inhibitors which mobilize leukaemic cells out of their protective niches by disrupting the AML–stroma interactions. These agents may also inhibit the pro‐survival signals provided to the blasts Dysregulation of chromatin modifiers is a recurrent and sentinel event in oncogenesis. Strategies that target the recruitment and/or catalytic activity of these enzymes at chromatin represent an attractive therapeutic modality in leukaemia The HMAs 5‐Azacytidine (azacitidine) and its deoxy analogue 5‐aza‐2′‐deoxycytidine (decitabine) are the two most extensively studied DNMT inhibitors and are approved for clinical use in haematologic malignancies in the United States. The cytidine nucleoside analogue Azacitidine which, upon cellular uptake, is in part converted into Decitabine, confers its cytotoxic effects via RNA and DNA incorporation, thereby disrupting protein and nucleic acid synthesis. DNMT inhibitors have been shown to induce response rates of 30% and more importantly prolong survival in elderly patients with AML in comparison with best available therapy for older patients IDH1 and IDH2), each seen in 8–12% of AML cases result in an abnormal oncometabolite 2‐hydroxyglutarate, which leads to a hypermethylated genome with a resultant block in differentiation IDH1 and/or IDH2 mutations, in combination with intensive chemotherapy, as well as with azacitidine in unfit patients. The 9.3‐month overall survival is also quite impressive in a pre‐treated population considering the expected 3‐month median survival in these patients IDH‐mutant AML patients. Enasidenib has recently been approved by the FDA for the management of relapsed/refractory AML in patients with IDH2 mutations.Neomorphic mutations in isocitrate dehydrogenase  produced complete remissions in only 2 of 34 patients with an MLL‐rearranged leukaemia The BET bromodomains are transcriptional coactivators involved in chromatin‐dependent signal transduction from master regulatory transcription factors to RNA polymerase II. The first direct‐acting bromodomain antagonist JQ1 was reported in 2010 DNMT3A and IDH2, are acquired early in leukaemic development are therefore present in nearly all clonal progeny and are almost always retained in AML at relapse NRAS and FLT3 that are acquired late in AML pathogenesis and often lost at relapse in vitro systems and in vivo xenograft models of AML has the potential to help guide the preclinical development of targeted agents that are effective in clinical trials as well as to understand mechanisms of therapy resistance. Recent improvements in the degree and scope of immunodeficiency as well as improved engraftment conditions have enabled more clinical specimens to be used in murine xenografts for preclinical testing NPM1 mutations in the absence of FLT3/ITD mutations in vitro and in vivo treatment systems with the potential to inform the assessment of the efficacy of novel agents in preclinical models. The primary drawback to MRD testing, however, is the uncertainty of which clonal hematopoietic cells are being measured. These methods detect residual disease but also measure aberrant pre‐leukaemic and non‐leukaemic hematopoietic cells which have unclear biological and prognostic significance One of the major challenges to the sustained efficacy of targeted therapy is the genomic and cellular heterogeneity of AML. While bulk disease at initial diagnosis is comprised of a small number of dominant clones Although the tremendous progress in genetic technologies has brought more insight into the pathobiology of AML, there is still a knowledge gap with regard to the most suitable targets. The reasons for his knowledge gap are multifaceted and include the complex molecular architecture of the disease with multiple driver mutations and interconnected signal transduction pathways The authors confirm that there are no conflicts of interest.


Nevertheless, our knowledge about the properties of ECC couplons is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle the fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion. CaCLEAN empowers the investigation of fundamental properties of ECC couplons in beating cardiomyocytes without pharmacological interventions. Upon examining individual ECC couplons at the nanoscopic level, we reveal their roles in the negative amplitude-frequency relationship and in β-adrenergic stimulation, including decreasing and increasing firing reliability, respectively. CaCLEAN combined with 3D confocal imaging of beating cardiomyocytes provides a functional 3D map of active ECC couplons . CaCLEAN will further enlighten the ECC-couplon-remodelling processes that underlie cardiac diseases.In beating cardiomyocytes, synchronized localized Ca This process is referred to as Ca2+-induced Ca2+ release (CICR)  and the functional state of RyRs  . It is ge (CICR) . For eace (CICR) . Many pr of RyRs .2+ transient and in cellular force production, fostering the mechanical output of the heart and thus providing a positive inotropic response   . The anaThe number and arrangement of LTCC and RyR clusters can be determined by appropriate immunohistochemistry. However, relating conclusions from such preparations to the physiological distribution of electrically evoked and functional ECC couplons  is rather difficult and often based on many assumptions. One of the most striking problems is that microscopic approaches to studying co-localization, even with super-resolution techniques, are still too coarse to identify functional coupling between these two partners reliably during normal ECC. In addition, fixation artefacts, such as shrinking of the preparation or alteration of the nanoscopic arrangement of proteins, might affect the aforementioned analysis. Thus, it clearly follows that approaches allowing for a robust in situ analysis  are eagerly anticipated not only to provide a full appreciation of the physiological 3D distribution of active ECC couplons but also to reveal pathophysiological alterations therein.2+ release clusters, that is Ca2+ sparks, can technically be monitored by laser-scanning microscopy . To this end, we performed model calculations of the putative fluorescence signal from an individual firing couplon and varied its distance to the plane of focus . The firing probability of ECC couplons across four consecutive Ca2+ transients demonstrated that only approximately 10% of couplons were always active  and 310 ± 146  . In addition to the changes in ECC couplon density, isoproterenol promoted the released Ca2+ content of each active couplon at low stimulation frequencies by more than 3-fold , the average number of active ECC couplons in a typical rat ventricular myocyte was approximately 16,500 couplons. This value correlates well with previously reported values  map of active ECC couplons to reconstruct their spatial distribution in an intact ventricular myocyte. To this end, we followed the principle protocol detailed in d values .2+ transients. These Ca2+ signals are the result of brief Ca2+ influxes during the cellular action potential and of the subsequent triggered Ca2+ release from the SR at sites of LTCC-RyR couplings, the so-called ECC couplons  the speed of the Ca2+ release process itself (in the millisecond range stimulation including a decreasing and increasing firing reliability, respectively.”We agree with the reviewers that it would be more comprehensive to provide additional details on this rather important and interesting aspect of our study in the Abstract. According to


Compelling evidences have demonstrated that Ca2+ might also enter through other cationic channels such as Store-Operated Ca2+ Channels (SOCCs), involved in several physiological functions and pathological conditions. The opening of SOCCs is regulated by the filling state of the intracellular Ca2+ store, the sarcoplasmic reticulum, which communicates to the plasma membrane channels through the Stromal Interaction Molecule 1/2 (STIM1/2) protein. In muscle cells, SOCCs can be mainly non-selective cation channels formed by Orai1 and other members of the Transient Receptor Potential-Canonical (TRPC) channels family, as well as highly selective Ca2+ Release-Activated Ca2+ (CRAC) channels, formed exclusively by subunits of Orai proteins likely organized in macromolecular complexes. This review summarizes the current knowledge of the complex role of Store Operated Calcium Entry (SOCE) pathways and related proteins in the function of cardiac, skeletal, and vascular smooth muscle cells.Cardiac, skeletal, and smooth muscle cells shared the common feature of contraction in response to different stimuli. Agonist-induced muscle's contraction is triggered by a cytosolic free Ca In 1990 Kwan and colleagues demonstrated that the refilling process does not involve a direct route to the intracellular stores, but it is the result of a sequential Ca2+ transport into the cytoplasm and the subsequent uptake from the stores by Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA)  and its mechanism of activation, this Ca2+ influx has been extensively investigated -evoked Ca2+ entry  syndrome, Orai1 was identified as a pore-forming subunit of SOCC , a Ca2+ sensor located in the cytoplasm that modulates STIM1–Orai1 complexes  were suggested as auxiliary components that co-activate and mediate STIM1–Orai1 interaction : the Ca2+ Release-Activated Ca2+ (CRAC) channel formed by homo- or hetero-multimeric Orai subunits (Orai1/2)  are non-selective for Ca2+ ions with a linear I/V relationship   from Patients with idiopathic Pulmonary Arterial Hypertension (iPAH). STIM2 might play a key role, but is not sufficient, in the upregulation of SOCE and PASMCs proliferation during iPAH  channel to reduce agonist-induced membrane depolarization, preventing excessive contraction of VSMCs  entry -regulated Ca2+ entry in VSMCs isolated from aorta  coupling to fulfill skeletal muscle's contraction  induced the depletion of SR which activates SOCE in isolated muscle fibers from adult mice  , have alterations in Ca2+-handling proteins involving STIM1 upregulation  located in the PM  are present in neonatal and adult cardiomyocytes  have been described in heart (Sabourin et al., 2+ signals required for the regulation of diverse cellular functions such as vascular reactivity, myotubes differentiation, skeletal muscle fatigue and endurance, cardiac homeostasis and pacemaking as illustrated in Figures 2+ signaling pathways used for the control of specific physiological and pathophysiological functions.Substantial progress has been made in terms of understanding molecular structures and mechanisms underlying SOCE in vascular, skeletal and cardiac muscle. Compelling experimental evidences have shown that SOCE plays a central role in providing the complex spatiotemporal CaConsequently, given the recent advances, the singular implication of these channels in muscle physiopathology is actually considered of a major interest. Nevertheless, more studies are needed to clarify their possible use as therapeutic target for the prevention of different muscle diseases.JA-M, IM-G, AD-R, IG-C, and TS wrote different parts of the manuscript. JAR, JR, AO, and TS discuss the manuscript. TS edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, only few data are available focusing on the first seconds and minutes of Ca2+ microdomain formation and related signaling pathways in activated T-lymphocytes. In this review, we condense current knowledge on Ca2+ microdomain formation in T-lymphocytes and early Ca2+ signaling, function of Ca2+ microdomains, and microdomain organization. Interestingly, considering the first seconds of T cell activation, a triphasic Ca2+ signal is becoming apparent: (i) initial Ca2+ microdomains occurring in the first second of T cell activation, (ii) amplification of Ca2+ microdomains by recruitment of further channels in the next 5–10 s, and (iii) a transition to global Ca2+ increase. Apparently, the second messenger nicotinic acid adenine dinucleotide phosphate is the first second messenger involved in initiation of Ca2+ microdomains. Ryanodine receptors type 1 act as initial Ca2+ release channels in CD4+ T-lymphocytes. Regarding the temporal correlation of Ca2+ microdomains with other molecular events of T cell activation, T cell receptor-dependent microdomain organization of signaling molecules Grb2 and Src homology [SH2] domain-containing leukocyte protein of 65 kDa was observed within the first 20 s. In addition, fast cytoskeletal changes are initiated. Furthermore, the involvement of additional Ca2+ channels and organelles, such as the Ca2+ buffering mitochondria, is discussed. Future research developments will comprise analysis of the causal relation between these temporally coordinated signaling events. Taken together, high-resolution Ca2+ imaging techniques applied to T cell activation in the past years paved the way to detailed molecular understanding of initial Ca2+ signaling mechanisms in non-excitable cells.Early Ca How Ca2+ signals are converted into meaningful cell responses has been a major area of interest in the past years  is essential for activation of T-lymphocytes, initiating transcriptional regulation, proliferation, and differentiation into effector T-lymphocytes. Activation is accompanied by a decrease in T-lymphocyte motility as well as to rounding up of the cell, thereby enabling immune synapse formation. A major regulator for transcriptional regulation, proliferation, and differentiation is Ca2+-dependent activation of calcineurin, and dephosphorylation of nuclear factor of activated T cells (NFAT) and its transport into the nucleus, where NFAT acts as one of the main transcription factors of T cells i. The increase of the luminal Ca2+ concentration then inactivates CRAC. Accordingly, the central role of PMCA and SERCA in the modulation of Ca2+ microdomain dynamics can be anticipated, though experimental data have not yet been published.Cas (CRAC) , therebytivation . An accu synapse , 24. Mac(d)2+ buffers: There are plenty of cytosolic and luminal Ca2+-binding proteins exerting Ca2+ buffering activity, thereby modulating spatiotemporal propagation of Ca2+ signals. In studies, modeling Ca2+ signaling in T-lymphocytes an intracellular concentration of Ca2+ buffers of 100 µM is presumed  in T cells largely abolished NAADP-evoked Ca2+ microdomains 2+ microdomains are essential for the induction of a sustained, global Ca2+ increase via activation of further channels.Ca2+ microdomains is that spatially restricted Ca2+ release in a trigger zone initiates and modulates the global Ca2+ increase. This was suggested by the so called two pool model: initial Ca2+ microdomains evoked by NAADP via RyR are then amplified by Ca2+-induced Ca2+ release (CICR) via RyR and IP3R   , 34 2+ microdomains in T-lymphocytes may not only result from NAADP activity but also may induce NAADP (and/or other second messenger) formation. This has been previously observed in germ cells 2+ signals leading to actin and signaling protein reassembly in the synapse , a transmembrane protein centrally involved in propagation of TCR signaling upon Zap70 phosphorylation and formation of diacylglycerol were observed as faster events within 4 s. PhosphoLAT then induces cytoskeletal changes: reorientation of microtubule-organizing center, followed by visible alterations of tubulin reorientation within 20 s 2+ signals drive exocytosis of cytolytic granules , it was postulated that local, NAADP-dependent Cagranules . In CTL,ctivated . In CTLsctivated .].(e)2+ microdomains generated by CRAC opening may differentially activate transcription factors as shown for c-fos and NFAT subtypes, which are both activated via Ca2+ signaling. Under regulation of caveolin-1, Ca2+ microdomains formed by CRAC opening may not only activate NFAT-mediated gene expression but also reduce c-fos-mediated transcription in RBL-1 mast cells   . These p3 cells) , 46.Accordingly, understanding the mechanisms underlying formation of Ca2+ microdomains consists of three phases: (i) initial Ca2+ microdomains occurring in the first second of T cell activation, (ii) recruitment of further channels within the next 5–10 s, and (iii) transition to global Ca2+ signaling by massive Ca2+ entry with tens of seconds  and (ii), too , too .2+ microdomains in activated T-lymphocytes as well as characteristics and potential function of Ca2+ microdomains is delineated in Figure Propagation of Ca2+ microdomains were analyzed for the first time in 2003 .T-lymphocyte Ca in 2003 . In Jurk2+ microdomains close to the plasma membrane were decreased upon cell activation with soluble anti-CD3. Interestingly, upon cADPR microinjection and usage of a cell-permeable cADPR derivative (cIDPRE) in a RyR knockdown Jurkat clone, diminished Ca2+ microdomains were observed , Caobserved . Similarphocytes . Furtherormation .3 into Jurkat T-lymphocytes gave important insights into the mechanism underlying initial Ca2+ microdomains , we were able to support these data in primary mouse RyR1membrane . These mfindings ] and a s2+ signals together with NAADP may be of central importance for initiation of exocytosis, while a global [Ca2+]i increase requires protein kinase C and ERK activation. Interestingly, treatment with the Ca2+-ATPase inhibitor CPA resulted in an abrogation of Ca2+ signals, suggesting that ER Ca2+ signals are prerequisites (not contributors!) for initial Ca2+ signals also in CTL  Ca2+ release from the ER (approximately 8 s after SLP65 recruitment). Interestingly, the Ca2+ signal started to spread from the central region of the cell, and simultaneously [Ca2+] increased in mitochondria, suggesting Ca2+ buffering. In addition, [Ca2+] also increased in Golgi system, which may play a role in vesicle trafficking during later stages of Ca2+ signaling  contributes to TCR-induced Came scale .2+) microdomains involved in T cell signaling. With a focus on the initial 10 s upon T cell activation, the following processes are described in the literature.Several studies point to the importance of characteristic  of 6.5 ± 0.5 s following photorelease of an antigenic peptide presented by an antigen-presenting cell (APC) was observed   . Further2 μm2/s) . Accordi2+ signal formation. Present data suggest that NAADP is the driving second messenger in early Ca2+ microdomain formation in T-lymphocytes and other immune cells. Possibly, NAADP formation or its binding to an unidentified accessory binding protein may be dependent on initial Ca2+ signals occurring in the first second of TCR activation. Interestingly, the NAADP target receptor RyR1 gives rise to initial Ca2+ microdomains (first 10 s of cell activation). By contrast, TPC apparently contribute to Ca2+ microdomain signaling in CTL, however, spatiotemporal data for TPC activation in primary CD4+ T-lymphocytes are still missing. To date, there is no indication that other channels such as TRPM2 or TRPV1 are involved in early (NAADP-mediated) Ca2+ microdomain formation by Ca2+ release from intracellular stores. Mitochondria apparently may play a central role in buffering Ca2+ microdomains in the immune synapse to prevent inactivation of Orai1.Table 2+ signaling events can be determined with high spatiotemporal resolution, while for other well-known T cell activation events, such as tyrosine phosphorylation, advanced methods are required. Nevertheless, the chronological sequence presented here may stimulate new approaches to understand the interactions between the different signaling modules involved in T cell activation.Taken together, presently CaBoth authors (IW and AG) wrote and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we show that the Ca2+ protein α1D of CaV1.3 channel is overexpressed in colorectal cancer biopsies compared to normal tissues. Gene silencing experiments targeting α1D reduced the migration and the basal cytosolic Ca2+ concentration of HCT116 colon cancer cell line and modified the cytosolic Ca2+ oscillations induced by the sodium/calcium exchanger NCX1/3 working in its reverse mode. Interestingly, NCX1/3 regulated membrane potential of HCT116 cells only when α1D was silenced, and blocking NCX1/3 increased cytosolic Ca2+ concentration and cell migration. However, membrane depolarization did not induce an increase in intracellular Ca2+. Patch-clamp experiments clearly showed that the inward Ca2+ current was absent. Finally, flow cytometry and immunofluorescence studies showed that α1D protein was localized at the plasma membrane, in cytosol and cell nuclei. Altogether, we uncover a novel signaling pathway showing that α1D is involved in the regulation of Ca2+ homeostasis and cell migration by a mechanism independent of its plasma membrane canonical function but that involved plasma membrane Na+/Ca2+ exchanger.It is generally accepted that voltage-gated Ca CaV are structurally made of five subunits  with the α1 subunit forming the pore of the channel1. The amino acid sequence is organized in four repeated domains each containing six transmembrane segments (S1–S6), and a membrane-associated loop between transmembrane segments S5 and S61. It is generally accepted that CaV control Ca2+ homeostasis of excitable cells  whereas non voltage-gated Ca2+ channels regulate Ca2+ homeostasis of non-excitable cells. Among CaV, CaV1.3 was found to be required for hearing2, for controlling excitability of chromaffin cells3 and for cardiac excitability by contributing either to diastolic depolarization of sino-atrial node pacemaker cells4 and atrial excitability5. In pathological conditions, CaV1.3 was found to contribute to the death of dopaminergic neurons in patients with Parkinson’s disease6 and to primary aldosteronism leading to arterial hypertension7.Voltage-gated Ca8. This was the case of the CACNA1D gene coding for the α1D subunit of CaV1.3 which is overexpressed in prostate, uterus and colon cancer8. In parallel, the α1D subunit was found to control the migration and the proliferation of endometrial cancer cells via the regulation of its expression by estrogens9 and the expression of androgens receptor in the prostate cancer cells10. Nevertheless, the biological role of the α1D subunit in colon cancer cells is not known, and it is important to recognize that it overexpression in colon cancer does not represent a causal link between high levels of the α1D protein and colon cancer. Colorectal cancer (CRC) represents major problems of public health because of its incidence and its mortality12. CRC is the third most common cancer in men  and the second in women  worldwide13. In France CRC is the second leading cause of cancer death and it accounts for nearly 12% of all cancer deaths, especially among those 65 years and older (http://www.e-cancer.fr/). In addition, CRC is characterized by metastasis development, which is the major cause of death of the patients developing a cancer. Although the mechanisms implied in the metastatic process are not yet completely elucidated, it is clear that the degradation of the extracellular matrix and the cellular migration, both regulated by Ca2+ channels14, play a pivotal role in this process.Recently, meta-analyses showed that expression of genes encoding CaV subunits are increased in various cancers15). Indeed, α1D protein does not only control the activity of the Ca2+-activated K+ channel, SK2, of atrial cells but also its expression and its membrane localization. The C terminus of α1D protein translocates to the nucleus where it functions as a transcriptional regulator to modulate the function of SK2 channel10. In addition, the alternative splicing of C terminus of α1D protein, besides modifying the activity of CaV1.3, affects the pharmacological properties of CaV1.3 and its sensitivity to the DHP16.It has not been determined if the regulation of the biology of cancer cells by α1D protein subunit depends on its plasma membrane canonical function. Indeed, α1D protein of CaV1.3 also has non-canonical functions and is involved in transcriptional regulation of the expression of other proteins including potassium channels  Ca2+ release.Here we investigated the role of the α1D protein of CaV1.3 in the migration of the non-excitable and epithelial cancer cells HCT116, its contribution in intracellular Ca17; www.proteinatlas.org). Among all α1 proteins only α1D was found overexpressed in CRC tissues compared to healthy tissues. Immunohistochemistry assays were performed on a series of 200 tissue-microarrays (TMA) included formalin-fixed and paraffin-embedded samples with paired tumor areas and healthy mucosa. α1D staining was stronger in adenoma and adenocarcinoma tissues compared to normal tissues and in adenoma tissues compared to adenocarcinoma tissues  ones , α1C (α subunit of CaV1.2), α1D (α subunit of CaV1.3) and α1F (α subunit of CaV1.4) in CRC patients using the Human Protein Atlas  decreased the number of migrating cells (transwell assay) by 50% and the viability of HCT116 cells by 25%  was added (see methods). Figure 2+ current was obtained in perforated-patch configuration, a configuration known to avoid rundown of the inward Ca2+ current  in 12 other cells. To test whether currents could be measured only in migrating cells, a cell monolayer in culture dishes was scratched, the migration of cells to the cleared area was inspected under a microscope 15 h after and cells were patched. Cell migration did not appear to affect the presence of Ca2+ current. Indeed, no inward Ca2+ current was recorded in migrating cells  was applied to cells. In this condition no inward Ca2+ current was observed . It was observed that the β3 subunit of CaV favors Ca2+ channel plasma membrane expression by facilitating the intracellular trafficking of α1D subunit toward the plasma membrane19. We observed a marked expression of the β3 subunit in HCT116 cells suggesting that its presence does not obligatory cause the plasma membrane localization of α1D protein  did not increase [Ca2+]c  a small steady-state inactivation of CaV1.3 channel had been observed21. To assay whether the absence of effect of elevated K+ concentrations on [Ca2+]c was due to the inactivation of α1D protein we first hyperpolarized plasma membrane of HCT116 using the SK3 channel activator, CyPPA23, before increasing external K+ concentration. Figure 2+]c. In addition, 10 µM verapamil did not change [Ca2+]c, as recorded in cells exposed to 40 mM of external K+  Ca2+ responses of HCT116 cells by decreasing the rising TG slope, TG area, TG peak Ca2+ responses and the relaxation TG slope  could be explained by regulation of PMCA Ca2+ efflux, Na+/Ca2+ exchanger (NCX) Ca2+ efflux or mitochondria Ca2+ uptake. Interestingly, silencing α1D had no effect on store operated Ca2+ entry (SOCE) of HCT116 cells  Na+ current drives Ca2+ extrusion from the cell while the reduction of Na+ current  forces Ca2+ entry by NCX thus working in its reverse mode. Figure 2+]c demonstrating that NCX drives Ca2+ extrusion of HCT116 cells. Western blot analysis of HTC116 extracts revealed a band of 110 kDa approximately, for NCX1 exchanger and bands of 110 to 140 kDa as expected for NCX3 exchanger  α1D reduced its activity and hyperpolarized cells to −57 mV. We next examined the ability of decreasing external Na+ concentration to hyperpolarize the cells. Figure + (Na10) solutions with a significant higher hyperpolarization membrane potentials observed in cells with silenced α1D. Finally, SEA0400 markedly increased the migration of HCT116 suggesting that α1D protein promotes cell migration by inhibiting NCX1/3  depolarization with high external K+ solutions did not increase the [Ca2+]c in contrast to what had been observed in excitable cells.Our data have demonstrated that α1D protein regulates migration/invasion of HCT116 cells mainly through its non-canonical activity because: i) we did not detect any voltage-gated inward Ca8 and has both canonical and non-canonical functions (for review see15). It was already observed that α1D protein controls migration and proliferation of endometrial cancer cells9. In these cells, α1D protein expression was regulated by estrogen controlling Ca2+ influx9, and nifedipine was found to decrease cell migration, proliferation and autophagy24. CACNA1D (gene encoding α1D) was found to be overexpressed in prostate cancer8 and nifedipine or verapamil reduced androgen-stimulated [Ca2+]c increase25. None of these reports have measured the Ca2+ channel activity of α1D protein using patch-clamp, the gold standard technique. Demonstrating that nifedipine/verapamil or knocking down α1D modified [Ca2+]c and cell migration is not sufficient to conclude that α1D protein works as a Ca2+ channel. While diltiazem has no effect on cell migration, we found that nifedipine increased the migration capacity of HCT116 cells. Guo et al.26 reported that nifedipine stimulated the migration of cancer cells via the axis of miRNA-524-5p-BRI3–Erk pathway independently of its Ca2+ channel-blocking activity. Among non-selective effects of pharmacological inhibitors of CaV channels, nifedipine and verapamil were also found to inhibit potassium channels28 and we demonstrated that multidrug resistance blockers mimicked the effect of verapamil on cell migration.α1D protein of CaV channels has been found expressed in various cancer forms2+ channel-blocking activities. Moreover, CaV channels are activated on membrane depolarization and we found that increasing external potassium concentration up to 80 mM did not change [Ca2+]c. This was not due to an inactivated state of CaV1.3 since with CyPPA, a SK3 channel activator regulating membrane potential of HCT116, [Ca2+]c was not changed when increasing external potassium concentrations. All together, these data support a non-canonical function of α1D protein in the regulation of [Ca2+]c and calcium oscillations of HCT116 cells.All these data strongly suggest that the effects of verapamil and nifedipine on HCT116 cell migration are not related to their Ca2+]c29, with NCX1 and NCX3 found expressed in HCT116 colon cancer cells. At physiological extracellular concentration of Na+ (140 mM), NCX allows the outward flow of Ca2+ 29. This is what we observed in HCT116 cells with NCX1/3 working in their forward mode. α1D expression partly block NCX1/3, which can explain the relatively polarized values of membrane potential of these cells (NCX blockade by silencing α1D depolarized cells by 20 mV). Using two blockers of NCX (KB-R7943 and SEA0400) we demonstrated that NCX1/3 regulate [Ca2+]c of HCT116 by driving Ca2+ extrusion out of cell. Moreover, blocking of NCX with SEA0400 increased the migration of the HCT116, probably by increasing [Ca2+]c. After switching to a low external Na+ solution an increase of [Ca2+]c was observed with the generation of Ca2+ oscillations. This is explained by the exchanger working in its reverse mode allowing an inward flow of Ca2+, which increase of [Ca2+]c. In this reverse mode NCX1/3 hyperpolarized HCT116 cells. The suppression of α1D protein of CaV1.3 was found to reduced [Ca2+]c, TG area, TG peak Ca2+ responses and increased hyperpolarization, [Ca2+]c and oscillations amplitudes following reduction of extracellular Na+ concentration. This suggests that α1D protein negatively regulates NCX1/3 working in its forward (inhibits outward flow of Ca2+) and reverse mode (inward flow of Ca2+). Intracellular Ca2+ oscillations has been observed in epithelial cancer colon cells30 and were found to play important roles in carcinogenesis31 but to our knowledge this is the first report showing a regulation of intracellular Ca2+ oscillations by α1D protein and NCX1/3. The molecular mechanisms regulated by NCX1/3 and CaV1.3 involved in [Ca2+]c and Ca2+ oscillations remain to be elucidated but it seems unlikely that this is through the canonical function of plasma membrane CaV1.3. We postulate that α1D protein interacts directly or indirectly with NCX1/3 inducing the inhibition of NCX1/3. Such a direct interaction between NCX and transient receptor potential (TRP) was demonstrated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments in rat cardiac myocytes32. Moreover, interaction between α1D protein and NCX1/3 should be favored by their localization in caveolae and probably by the presence of specific protein and/or lipids in these lipid rafts like caveolin and cholesterol. Another possibility is that a particular non pore α1D protein, due to mutation or posttranslational modifications, may favor the interaction and the inhibition of NCX1/3. Moreover, α1D protein both from plasma membrane and ER can modulate plasma membrane NCX1/3. Interestingly, even with low external Na+ concentration, [Ca2+]c was found to be lower after knocking down of α1D compare to control cells. This can be explained by a control of ER Ca2+ release and/or PMCA Ca2+ efflux and/or mitochondria Ca2+ uptake by α1D. We can speculate that this depends on the localization of α1D that was mainly found in intracellular compartments and probably ER. The cause of this particular localization remains to be elucidated.α1D protein was found to regulate NCX of HCT116 cells. NCX genes comprise a family of three genes  thought to be involved in maintaining a [Ca2+]c through its non-canonical function α1D protein was found to work as a transcription factor regulating the expression of proteins involved in the regulation of [Ca2+]c and cell migration. Indeed, a fragment of the CaV1.3 C-terminus was reported to be translocate to the nucleus where it regulates the expression of the Ca2+-activated K+ channel, SK2 channel, and protein regulating cell migration such as the myosin light chain33. The C-terminus of CaV1.2 also acted as a transcription factor and its overexpression altered the expression of NCX1, the Ca2+ channel TRPV4 and Ca2+-activated K+ channel, SK3 channel34. In addition, the C-terminal end of CaV1.3 would play a significant role in the sensitivity to antagonists of CaV1.3 particularly those of the family of dihydropyridines (ex: nifedipine). Indeed, the alternate splicing of distal C-terminal end of CaV1.3, besides modifying the activity of CaV1.3, affects the pharmacological properties of CaV1.3 and the sensitivity to the DHP16. Since we detected α1D protein in the nucleus further work needs to be done to confirm this data and elucidate the role of nuclear α1D in HCT116 cells.In addition to regulate c was calculated as described previously using in situ calibration38. A Kd of 135 nM was used for these calculations, according to the supplier information on this batch of Fura-2 . The physiological saline solution (PSS) or 2Ca solution had the following composition (in mM): NaCl 140, MgCl2 1, KCl 4, CaCl2 2, D-glucose 11.1 and HEPES 10, adjusted to pH 7.4 with NaOH. The Ca2+ free solution or 0Ca is a PSS solution without CaCl2 and with 1 mM EGTA. The ionomycine was used at 5 µM. High external potassium solutions were prepared by adding 56 mM KCl (K60) or 76 mM KCl (K80) and high external solutions by adding 56 mM NaCl (Na196) or 76 mM NaCl (Na216).Samples were analyzed using either a microscope or a FlexStation. The excitation light source was a 75-W Xenon arc lamp. Excitation light at the two-excitation wavelengths maxima of Fura-2 (340/380 nm) was chopped by a monochromator . The excitation protocol was a 50 ms excitation at each wavelength every 4 s. Excitation light was directed through a 20× objective with a numerical aperture of 1.4 . Fluorescence emissions at 510 ± 20 nm were detected by a filter (PMT) placed in the microscope body. Cells were then added in 96-well clear-bottom plates , at a density of 20,000 cells/well. For the Flexstation, cells were incubated at 37 °C under 5% CO2+ TG responses was measured by calculating the difference between the basal and the maximal Ca2+ ratio after TG application, in Ca2+-free solution. The rising slope of the Ca2+ TG responses was determined by linear regression curve fitting 20 sec after TG application and was an estimation of the speed of the Ca2+ release by ER. The area of the Ca2+ TG responses was determined by integral calculation of the area under the curve, after TG application. The relaxation of the Ca2+ TG responses was determined by the half-return time: it is the subtraction of time at the peak minus the time the return at the half of peak value. Peaks and area TG Ca2+ responses represented both Ca2+ ER release and/or [Ca2+]c decrease  Ca2+ efflux or mitochondria Ca2+ uptake but not SERCA Ca2+ efflux since TG is present).The peak amplitude of the Ca22. Trans-well invasion were assessed as trans-well migration assays but membrane was covered with a Matrigel® matrix39. Briefly, after 24 h, stationary cells were removed from the top side of the membrane, whereas migrated cells in the bottom side of the inserts were fixed, stained, and counted in five different fields . At least three independent experiments were each performed in triplicate. Wound healing migration assays were performed from cell monolayer on 6-well culture plate where wounds were made with a sterile 2mm-wide tip. After wash, cells were treated or not with different drugs. Phase-contrast images of the wound were obtained at the time of scratching and after 12 h. Automatic acquisitions were performed on a Nikon microscope (eclipse ti), coupled to a Nikon camera (DS Qi2). The system includes a cage incubator  controlling temperature, levels of CO2 and O2. Analyses were performed using NIS Element AR software. Brieflly, the analyzes were processed by measuring the area of the injured area to 0 h and 12 h after injury with and without treatment. The injury area before and after the test was measured and compared to the control. The values were plotted as the percentage of the wound closure, with the initial width set to the normalized control.Trans-well migration assays were performed as described previouslyV1.3 channel, anti-CaV1.3 (extracellular) antibody  was used for intracellular and extracellular staining. For intracellular staining, cells were washed with cold PBS then incubated for 20 min with 100 μL of Cytofix/Cytoperm™Fixation/Permeabilization Kit (BD Biosciences). After, cells were incubated with a rabbit anti-CaV1.3 antibody for 45 min at 4 °C. Then, cells were washed and incubated with an anti-rabbit coupled to an Alexa Fluor 488 for 45 min at 4 °C. All flow cytometry (FCM) analyses were performed with a minimum of 10000 events using a Gallios flow Cytometer and Kaluza version 1.2 (Beckman Coulter).Cells were incubated at 4 °C with saturating concentrations of CaV1.3 antibody (1:200 Alomone ACC-311) in the dark for 45 min, washed twice with PBS, supplemented with 0.1% Azide-PBS-4% FBS. The same antibody directed against an extracellular epitope of Ca3 (extracellular) antibody  for 1 h at 4 °C. Glasses are washed (3x) and incubated with a secondary AlexaFluor 488-conjugated anti-rabbit . Control experiments were performed using only secondary AlexaFluor 488-conjugated anti-rabbit. Acquisitions were performed with a JAI camera (model CV-M4 + CL), with the use of an automated filter wheel coupled to a Leica DMRB fluorescence microscope (Leica Microsystems). Analyses were performed using ImageJ software .HCT116 cell line were plated overnight on glasses and subsequently fixed (PBS 1X PFA 4%), permeabilized (Triton × 100 0.1%), blocked (BSA 5%) and incubated with primary rabbit anti-CaV1.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplemental Method ans Figures