In addition, the application of in vivo imaging approaches has revealed that Ca2+ is fundamental to the synchronization of the neuronal networks that make up circadian pacemakers. Conversely, the activity of circadian clocks may influence Ca2+ signaling. For instance, several genes that encode Ca2+ channels and Ca2+-binding proteins display a rhythmic expression, and a disruption of this cycling affects circadian function, underscoring their reciprocal relationship. Here, we review recent advances in our understanding of how Ca2+ signaling both modulates and is modulated by circadian clocks, focusing on the regulatory mechanisms described in Drosophila and mice. In particular, we examine findings related to the oscillations in intracellular Ca2+ levels in circadian pacemakers and how they are regulated by canonical clock genes, neuropeptides, and light stimuli. In addition, we discuss how Ca2+ rhythms and their associated signaling pathways modulate clock gene expression at the transcriptional and post-translational levels. We also review evidence based on transcriptomic analyzes that suggests that mammalian Ca2+ channels and transporters  as well as Ca2+-binding proteins  show rhythmic expression in the central brain clock and in peripheral tissues such as the heart and skeletal muscles. Finally, we discuss how the discovery that Ca2+ signaling is regulated by the circadian clock could influence the efficacy of pharmacotherapy and the outcomes of clinical interventions.In animals, circadian clocks impose a daily rhythmicity to many behaviors and physiological processes. At the molecular level, circadian rhythms are driven by intracellular transcriptional/translational feedback loops (TTFL). Interestingly, emerging evidence indicates that they can also be modulated by multiple signaling pathways. Among these, Ca 2+ signaling and the circadian clock. In Drosophila and mice, circadian clocks impose a daily rhythmicity to Ca2+ signaling; and, conversely, Ca2+ rhythms and signaling contribute to transmitting daily external signals to the clock TTFL. This bidirectional regulation is critical to the daily rhythmicity of many physiological and behavioral processes.Reciprocal relationship between Ca In the cytoplasm, the corresponding PER and either TIM (Drosophila) or CRY  proteins dimerize, re-enter the nucleus, and inhibit the activity of CLOCK-CYCLE, thereby preventing their own expression. The exact functioning of this core TTFL is, in addition, modified by a growing number of intracellular factors, which include additional secondary TTFLs, kinases and phosphatases that regulate the stability of PER and TIM (CRY) proteins, as well as chromatin and chromatin-modifying complexes, all of which contribute to producing a molecular rhythm with near-24 h periodicity. Circadian clocks are classified according to where they reside: central oscillators are located in the brain whereas peripheral oscillators can be housed in a wide variety of tissues, where they impose a daily rhythmicity to the physiology of each organ . In addition, we discuss the mechanism by which Ca2+ signaling affects clock function , and, in turn, how clock function affects Ca2+ signaling (see section “Circadian Regulation of Ca2+ Associated Proteins”). Finally, as a result of our growing appreciation of the importance of clocks in health and disease, we discuss how our knowledge of the reciprocal relationship between clocks and Ca2+ signaling impacts the effectiveness of drugs and is relevant to the development of improved therapeutics .All multicellular animals contain a biological clock that allows them to anticipate the daily changes in the environment, such as the arrival of dawn or dusk. These are endogenous mechanisms that are cell-autonomous and are invariably controlled by highly conserved intracellular transcription-translation feedback loops (TTFL) . At theich organ . Yet, thch organ . At the ch organ . These s2+ levels ([Ca2+]i) are essential for the proper functioning of many cellular and physiological processes, including circadian rhythms. For example, in the case of pacemakers housed within neuronal tissues, fluctuations in [Ca2+]i have been associated with the circadian control of neuropeptide release, the expression of clock genes, synaptic plasticity, and the periodicity of rhythmic behaviors   , a peripheral clock that regulates the circadian rhythm of adult eclosion  . Remarka the SCN . In addi is weak , suggest is weak , are rel+ phases . In partchronous  and the chronous . In addieclosion . A studyhe brain , which ahe brain . Of notem the PG , suggest insects , they ha2+ oscillations in biological clocks, these extracellular signals are not the only relevant ones for regulating Ca2+ rhythms. In mice, an early study using FURA-2, a synthetic fluorescent Ca2+-sensitive dye, showed that Ca2+ oscillations are completely abolished by tetrodotoxin (TTX) treatments, suggesting that neuronal firing of SCN neurons is essential for maintaining these rhythms  display increased phase delays when they receive a light stimulus in the early part of the night , to set the phase of a group of E-cell neurons  in circadian pacemaker neurons, lengthen the periodicity of the circadian rhythms of locomotor activity and of adult eclosion  and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in rat liver explants lengthens the period of Per1-luc oscillations  regulation via Ca2+/cAMP-responsive elements (CREs); and by modifying the phosphorylation state of clock proteins that affect the functioning of TTFLs at (b) the transcriptional or (c) the post-translational level.The growing evidence of the role of Ca2+, into the rhythm of expression of clock genes. For instance, in the hamster SCN, the phase-shifts in Per1 and Per2 induced by light are mediated by CaMKII  reorganizes this hierarchical organization altering Ca2+ rhythms in clocks cells and lengthening the period of CRE-luc and Per-luc cycling in the SCN. These effects are not mediated through Gs or Gi, indicating that CRE-mediated transcription is exclusively activated through the Gq-Ca2+ axis.cAMP-responsive element is a sequence that binds CRE-binding protein (CREB) and is present within the promoter region of several mammalian clock genes . In fliey CaMKII , which cy CaMKII   and PER proteins in head extracts but not those of their respective transcripts. Of note, the effect of calcineurin on clock proteins levels is abolished when the proteasomal machinery is inhibited  also exhibit circadian regulation at the transcriptional level in the SCN   , suggestell line , support2+ influx from the extracellular milieu via voltage-gated Ca2+ channels (VGCCs). Indeed, in the SCN, some VGCCs subunits mediate the phase shifting induced by photic inputs as well as the daily changes in conductance  at ROREs and direct mutagenesis of this sequence abolishes the oscillation of Cacna1c mRNA levels, indicating that REV-ERBα plays a critical role in this circadian regulation. Of note, this mechanism is not the only one that has been associated with the rhythmic control of expression of L-type VGGCs. Indeed, in chicken cone photoreceptors, L-type-VGCCα1Cs display a diurnal rhythm of expression, which is controlled at the posttranscriptional level by microRNAs  , display a rhythmic expression in members of the PKC pathway , and in genes encoding Ca2+ channels  and genes involved in Ca2+ signaling   . Many ofineurin) . Howeverifferent  , the Ca2+ transporter of the sarcoplasmic reticulum (serca), and the Ca2+ buffer protein, parvalbumin, which did not occur in the muscles of CLOCK-knockout mice  is restricted to the early part of the night, which prevents it from binding to SERCA , which is characterized by mood instability and abnormalities in sleep and daily activity schedules  (2+ pathways including cacna1c (L-type VGCC) are risk alleles for BD  . In BD, ealtime) . In partealtime) . Additios for BD . Interes rhythms . But VGCtagonist . In agrepatients . Given tpatients , conside2+ channel subunits, which display a rhythmic expression in the heart . Consistent with this hypothesis, harmonizing the timing of administration of a drug with the time of peak expression of its target improves the effectiveness of treatments for cardiovascular disease, hypercholesterolemia, and obesity  (Considering the daily changes in Caon (I/R) . A recenon (I/R) . Intereson (I/R)  and, remon (I/R) . This tion (I/R) . Since t2+ signaling in biological clocks may have significant implications for medicine (2+ signaling into account in pharmacological therapies and medical interventions could improve drug efficacy and postoperative clinical outcomes, supporting the emerging idea that circadian biology should be an integral part of translational research (Collectively these findings provide strong evidence that the circadian control of Camedicine . Thus, tresearch .We currently have a very detailed understanding of how circadian clocks function. By comparison, much less is known about how they are synchronized and coordinated; how they transmit time information to the host; and, in turn, how they can be affected by the host’s physiology and behavior. All of these system-level functions ultimately converge on intracellular signals that affect gene expression, which, in turn, alter the activity of clocks, and the physiology of cells, organs, and organisms. Future work aimed at increasing our knowledge on how intracellular signaling regulates, and is regulated, by clocks will be important for furthering our understanding of how this intricate network of interactions is effected and how a unified time for the organism is maintained yet can be modulated by the host. It will also be critical for the development of more effective chronomedicine prescriptions aimed at improving the effectiveness of drugs while reducing their negative and off-target effects.JC-L wrote the first draft. JC-L and JE edited manuscript. Both authors contributed to the article and approved the submitted version.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, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules.Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca FAM38A gene) and Piezo2  on the direct mechanical stimulation-induced intracellular free Ca2+ concentration ([Ca2+]i) increases. In addition, we examined the role of Piezo1 channels during dentinogenesis using mineralizing assays.Odontoblasts have been reported to express Piezo1 and Piezo2 channels . In addi neurons . Odontob neurons . However2 for 48h. The odontoblast suspension was adjusted to a density of 5×104 cells/ml.Human odontoblast cells were obtained from a healthy third molar and immortalized by transfection with the human telomerase transcriptase gene . The res2, 0.5 MgCl2, 10 HEPES, 10 glucose, and 12 NaHCO3 (adjusted to pH 7.4 with Tris) was used as the extracellular solution. A selective pharmacological Piezo1 channel activator, Yoda1  containing (in mM) 136 NaCl, 5 KCl, 2.5 CaClr, Yoda1 , was obt and 0.1% (w/v) pluronic acid F-127 (Life Technologies). They were then washed with a fresh standard solution. A dish containing fura-2-loaded human odontoblasts was mounted on the stage of a microscope , which was installed with an HCImage system , an excitation wavelength selector, and an intensified charge-coupled device camera system. Fura-2 fluorescence emission was recorded at 510nm in response to alternating excitation wavelengths of 340nm (F340) and 380nm (F380). The intracellular free Ca2+ concentration ([Ca2+]i) was defined as the fluorescence ratio (RF340/F380) at two excitation wavelengths and then described as F/F0 units; the RF340/F380 value (F) was normalized to the resting value (F0). All the experiments were performed at room temperature (28°C). Standard ECS with or without pharmacological Piezo1 channel activator/inhibitors was applied by superfusion using a rapid gravity-fed perfusion system  and microscope (Olympus). The distance from a mechanically stimulated human odontoblast to each neighboring cell was determined from the images obtained (HCImage) by measuring the shortest distance between each pair of cells.To generate the odontoblasts transfected by shRNA, including vectors specific for human Piezo1 (shRNA-Piezo1 transfected cells) or an empty vector control (shRNA-Control transfected cells), the Lenti-X™ 293T cell line  was used to produce lentiviral vector particles. Packaging plasmids pCAG-HIVgp  and pCMV-VSV-G-RSV-Rev  were used for lentiviral vector packaging. Lentiviral vectors specific for human Piezo1 (Sigma-Aldrich) and a TRC2 empty vector control (Sigma-Aldrich) were used in this experiment. Viral supernatants were harvested after 72h. Human odontoblasts with 60–80% confluency were transfected with lentiviral vector particles including shRNA and then incubated under normal growth conditions for 72h.Human odontoblasts were transferred to eight-well glass chambers  and maintained under culture conditions as described above. Cells were fixed with 4% paraformaldehyde  and washed with 1× PBS . After 10min of incubation with blocking buffer  at room temperature, the following primary antibodies were applied for 6h: mouse monoclonal anti-dentin matrix protein 1 , mouse monoclonal anti-DSPP , or mouse monoclonal anti-nestin . The secondary antibody  was then applied for 1h. Human odontoblasts, which were transfected with shRNA-Piezo1 and shRNA-Control, were also transferred to eight-well glass chambers , maintained in culture conditions, and fixed with 4% paraformaldehyde . The cells were washed with 1× PBS (Thermo Fisher Scientific K.K.), and incubated for 10min with blocking buffer  at room temperature. Next, Alexa Fluor® 488 conjugated rabbit polyclonal anti-Piezo1  and Alexa Fluor® 594 conjugated rabbit polyclonal anti-Piezo2  were applied for 6h. Stained samples were mounted in mounting medium with 4,6-diamidino-2-phenylindole . Immunostaining was observed and analyzed, and photographs were taken (as 8-bit TIFF images) using a fluorescence microscope . For the negative control, the cells were incubated with non-immune antibodies diluted to equivalent concentrations to those of the primary antibodies (data not shown). For shRNA-Piezo1 transfected and shRNA-Control transfected cells, TIFF images obtained with 8-bit of 1,920×1,440 pixels were converted to reversed grayscale. Then, the percentage area of the Piezo1- or Piezo2-immunopositive cells was measured using automated thresholds of ImageJ software .2. To determine the effects of Piezo1 activity on mineralization, the cells were cultured in mineralization medium without (as control) or with the pharmacological Piezo1 modifier, Yoda1 (2μM), Dooku1 (10μM), or Yoda1 (2μM) and Dooku1 (10μM) for 28days. The odontoblasts transfected with shRNA, including vectors specific for human Piezo1, or including an empty vector control (see above), were also grown to full confluency in basal medium, and then transferred to mineralization medium at 37°C in 5% CO2 for 28days. During the 28-day culture period, mineralization media with or without pharmacological agents were changed twice a week  of Alizarin red staining were normalized to the mean intensity values of areas without cells (I0).Human odontoblasts were grown to full confluency in basal medium, and then transferred to mineralization medium  for growth at 37°C in 5% COe a week . To deteN observations, where N represents the number of experiments or cells tested. The Kruskal-Wallis test, Friedman test, and Dunn’s post-hoc test were used to determine non-parametric statistical significance. Parametric statistical significance was determined using a two-tailed Student’s t-test to analyze the percentage areas of Piezo1/2 channel-immunopositive cells in the immunofluorescence analyses. Statistical significance was set at p<0.05. Statistical analysis was performed using GraphPad Prism 5.0 .Data are expressed as the mean±SE or SD of the mean of 2+]i was measured. When we applied mechanical stimulations to single human odontoblasts using a glass micropipette moving vertically (8μm) in a downward direction from a position just above the surface , we observed transient increases in [Ca2+]i in the mechanically stimulated human odontoblasts  to neighboring odontoblasts (red circles). The peak values of [Ca2+]i were 2.52±1.45 (N=9), 1.59±0.55 (N=15), 1.34±0.32 (N=8), 1.07±0.03 (N=4), 1.11±0.09 (N=6), 1.06±0.03 (N=6), and 1.02±0.01 (N=5) F/F0 units in human odontoblasts located within distances of 0–10μm, 11–20μm, 21–30μm, 31–40μm, 41–50μm, 51–60μm, and 61–70μm from mechanically stimulated human odontoblasts, respectively , the application of 2μM Yoda1 to human odontoblasts showed a rapid and transient [Ca2+]i increase  in F/F0 units (F/F0=1). Yoda1 induced-[Ca2+]i increases were significantly and reversibly inhibited by extracellular 1μM Gd3+, a non-selective Piezo1 channel inhibitor to 1.36±0.17  in F/F0 units, reversibly  in F/F0 units (N=6) in 3+ to 1.47±0.31  in F/F0 units  after Gd3+ and to 1.81±0.41 (N=6) after Dooku1 application  and shRNA-Control transfected cells  of the stains were normalized to the mean intensities of adjacent areas without cells  activates ionotropic ATP receptor subtype 3 (P2X3 receptors) in the intradental Aδ neurons. The activation of P2X3 receptors induces an action potential in pulpal Aδ neurons, indicative of the generation of dentinal sensitivity  application on dentin; In line with our results, it has been described that Piezo1 channels are expressed not only in multipotent stem cells from human exfoliated deciduous teeth (SHED), but also in odontoblast processes . In addi3+ (1μM) and Dooku1 (10μM) almost completely suppressed direct mechanical stimulation-induced [Ca2+]i increases in single human odontoblasts. We also only observed small residual responses in mechanical stimulation-induced [Ca2+]i increases in shRNA-Piezo1 transfected cells compared to those in the shRNA-Control transfected cells. Therefore, together with the present results, Piezo1 channels may act upstream of mechanosensitive cation channels, including TRPV1, TRPV2, TRPV4, and TRPA1 channels, as well as Piezo2 channels, in odontoblasts.Recent evidence have indicated that mouse odontoblasts also express Piezo2 channels . In line2+]i increases mediate the intercellular signaling networks among them. Piezo1 channels predominantly contribute to the detection of cellular deformation, such as mechanical stimulation, induced by dentinal fluid movements in dentinal tubules, during various stimuli applied to the exposed human dentin surface, such as enamel lesions including dental caries and/or acid erosion. However, Piezo1 channel activity negatively regulates not only reactionary dentinogenesis, but also physiological/developmental dentinogenesis. This suggests that Piezo1 channel activity may physiologically prevent the lifelong loss of volume in the pulp chamber by excessive dentin formation induced by mechanical stimuli applied on the teeth, such as mastication.In conclusion, we demonstrated that human odontoblasts functionally express Piezo1 channels, and mechanical stimulation-induced-[CaThe original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.MM, MK, TI, and YS conceived and designed the experiments. MM, MK, TO, TN, SO, MA, SN, and TA performed the experiments. MM, MK, TO, TN, SO, MA, SN, TA, TI, and YS were responsible for analyzing and interpreting the data and drafting and critically revising the intellectual content of the article. TI and YS were responsible for the final approval of the version to be submitted or published. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and have been confirmed to be critical in tumorigenesis and to be potential biomarkers or therapeutic targets. However, only a few circRNAs have been functionally characterized in pediatric acute myeloid leukemia (AML).Here, we investigated the expression pattern of circRNAs in pediatric AML using a circRNA microarray. The characteristics, potential diagnostic value, and prognostic significance of circRNF220 were evaluated. A series of functional experiments were performed to investigate the role of circRNF220 in primary pediatric AML cells. Then we investigated the aberrant transcriptional networks regulated by circRNF220 in primary AML cells by RNA-seq. Furthermore, biotin RNA pulldown assays were implemented to verify the relationship between circRNF220 and miR-30a.We identified a circRNA, circRNF220, which was specifically abundant in and accumulated in the peripheral blood and bone marrow of pediatric patients with AML. It could distinguish AML from ALL and other hematological malignancies with high sensitivity and specificity. Significantly, circRNF220 expression independently predicted prognosis, while high expression of circRNF220 was an unfavorable prognostic marker for relapse. Furthermore, we characterized the function of circRNF220 and found that circRNF220 knockdown specifically inhibited proliferation and promoted apoptosis in AML cell lines and primary cells. Mechanistically, circRNF220 may act as an endogenous sponge of miR-30a to sequester miR-30a and inhibit its activity, which increases the expression of its targets MYSM1 and IER2 and implicated in AML relapse.Collectively, these findings demonstrated that circRNF220 could be highly efficient and specific for the accurate diagnosis of pediatric AML, with implications for relapse prediction.The online version contains supplementary material available at 10.1186/s12943-021-01395-7. Acute myeloid leukemia (AML) is a relatively rare malignancy among childhood malignancies, accounting for less than one-fifth of acute leukemia cases diagnosed in childhood. However, it remains a challenging disease with a poor outcome compared with that of pediatric acute lymphoblastic leukemia (ALL) . DespiteCircular RNAs (circRNAs) are covalently closed nonlinear RNA molecules that are produced from pre-mRNA backsplicing and have been considered faulty splicing products for several decades . Howeverin vitro and in vivo [Emerging evidence has shown that the roles of a few circRNAs are well established in AML . For ins in vivo . It is wHere, we sought to investigate the regulatory roles played by circRNAs in pediatric AML. Our screening strategy identified that circRNF220, a circRNA derived from the RING domain E3 ubiquitin ligase gene RNF220, was specifically upregulated in pediatric AML and was required for AML progression. CircRNF220 KD significantly impeded the activities of primary pediatric AML cells. Furthermore, we revealed that circRNF220 performed its regulatory functions as a competing endogenous RNA by binding miR-30a and then regulating the expression of downstream genes Myb like, SWIRM and MPN domains 1(MYSM1) and immediate early response 2(IER2). Importantly, we showed that circRNF220 not only outperformed current markers in terms of its high sensitivity, specificity and diagnostic simplicity but also demonstrated prognostic value for early recurrence by evaluation in bone marrow (BM) and even peripheral blood (PB) samples.Diagnostic BM or PB samples were collected from 149 pediatric patients (1 to 14 years of age) with de novo AML of French-American-British (FAB) classification M1-M7 enrolled in this study between January 2015 and May 2020 at the Guangzhou Women and Children’s Medical Center of Guangzhou Medical University who had available pretreatment and post chemotherapy cell samples. The demographic characteristics of the AML patients are summarized in supplementary Table SP-value < 0.05 were identified as significantly differentially expressed circRNAs. We further confirmed the microarray results for 10 randomly selected circRNAs by qRT-PCR in samples from 10 newly diagnosed AML patients and healthy controls.A circRNA microarray  containing more than 5000 probes specific for splice sites in human circRNAs was used in this study. After hybridization, 5 pediatric AML samples (pooled) and 5 healthy donor BM samples (pooled) were examined using the circRNA microarray provided by Kangcheng Bio-Tech Inc. R software was used to process the subsequent data after quantile normalization. Differentially expressed circRNAs were identified through volcano plot filtering and fold change filtering. CircRNAs with a fold change ≥ 2.0 or ≤ 0.5 and a Ct−△△ method, and GAPDH was used as the internal standard. All assays were performed in triplicate. The sequences of all primers used are available . To quantify the amounts of mRNAs and circRNAs, cDNA was synthesized from 500 ng of RNA with PrimeScript RT Master Mix . QPCR analyses were performed using SYBR Premix Ex Taq II (Takara) and a LightCycler® 480 Real-Time PCR System . Specifically, divergent primers annealing at each side of the backsplice junction were used to determine the abundances of circRNAs. The expression levels were calculated using the 2RNA FISH was performed on AML cells and 293 T cells using an RNA Fluorescence In Situ Hybridization Kit  in accordance with the manufacturer’s protocol. Probe sequences designed by Exonbio Lab are listed in Supplementary Table S2, and all cell lines were routinely tested for mycoplasma contamination. AML BM mononuclear cells were prepared by Ficoll density centrifugation at 600 × g for 30 min, and then were thawed and plated in MEMα (Gibco) supplemented with 10% FBS, 20 ng/ml hTPO, 20 ng/ml hIL-3, 20 ng/ml hG-CSF, and 1 × penicillin/streptomycin.Human leukemia cell lines  and the human embryonic kidney cell line HEK-293 T were purchased from the American Type Culture Collection and maintained in RPMI 1640 medium or DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in 5.0% COAML cells were exposed to 2 μg/mL actinomycin D  to block transcription for 4, 8, 12, 24, and 48 h. Then, the cells were harvested, and the stability of the circRNF220 and RNF220 mRNAs was analyzed using qRT-PCR.All in vitro studies were repeated three times in AML cell lines, or performed in more than three primary AML cases BM cells.Total RNA was treated with RNase R. Briefly, 5 μg of total RNA was exposed to 2 U/μg RNase R (Epicenter Biotechnologies) at 37 °C for 30 min, and RNase-free water was used as a control (Mock). Digested RNA was subsequently purified using an RNeasy MinElute Cleanup Kit (Qiagen). The RNA concentrations of the treated samples were determined, and 1 μg of treated RNA was used for qRT-PCR.The nuclear and cytoplasmic fractions were extracted according to the manufacturer’s protocol using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). The effectiveness of nuclear and cytoplasmic separation was assessed by determining the protein levels of histone H3 and GAPDH, which are specifically expressed in the nucleus and cytoplasm, respectively.Small interfering RNAs (siRNAs) against circRNF220 (siRNA-circRNF220) and the negative control RNA duplex (siRNA-NC) were purchased from RiboBio, Guangzhou, China. The miR-30a mimics (miR-30a) or its inhibitor (inhibitor-miR-30a) and scrambled oligonucleotides (miR-NC or inhibitor-NC) were purchased from GenePharma Biotech .The plasmid for circRNA overexpression (OE) was constructed using the pcDNA3.1( +) circRNA Mini Vector, which was produced by the Forevergen Company . The region spanning the second exon of the RNF220 gene that constitutes the circRNA was PCR amplified from cDNA and seamlessly cloned between the splicing signals AG and GT, which are surrounded by the minimal introns that facilitate the generation of circRNF220 (pcDNA3.1-circRNF220). The construct was verified by sequencing.5 cells/sample), which were transfected with 100 pmol of the siRNAs in T buffer using the Neon® Transfection System (Invitrogen) with settings of 1400 V, 10 ms and 1 pulse.In addition, based on the expression levels of circRNF220 in AML cells, we selected the AML cell lines with comparatively low circRNF220 expression  to perform gain-of-function experiments. Primary BM cells expressing comparatively high levels of circRNF220 were used to perform loss-of-function experiments. Unless otherwise noted, the plasmids and siRNAs were delivered into cultured AML cells by electroporation as we reported before . The excThe transfected cells were collected at different time points after electroporation for biological functional analyses and for RNA extraction.AgeI and EcoRI sites in the 5’LTR of MCS-CBh-gcGFP, and the integrity of the insert was confirmed by sequencing.The circRNF220 KD lentivirus (LV-sh-circRNF220) was produced by GeneChem Company . The shRNAs only specifically targeted the head-to-head splicing junction of circRNF220. The particular sequences were synthesized and cloned into the AML BM cells were transduced with retroviral vectors according to the protocol. Briefly, LV-sh-circRNF220 lentiviral particles were centrifuged for 2 h at 1,200 × g in 24-well plates precoated with 100 μg/ml RetroNectin . Three days later, the cells were sorted to separate GFP + populations for subsequent experimental studies.Cell proliferation was assessed using a Cell Counting Kit-8 , and samples were evaluated in a microplate reader  as described previously .Treated AML cells were stained with PI  containing RNase A for 30 min at 37 °C. Cells were analyzed for DNA content by fluorescence activated cell sorting (FACS)  with ModFit software used for data analysis, as described previously .After treatment with 25 ng/mL phorbol-12-myristate 13-acetate  for 2 days, THP-1 cells were reseeded in fresh medium without PMA for 2 more days to allow cell recovery. Then, monocytic differentiation was evaluated based on the cell morphology on Wright-Giemsa-stained slides and by FACS analysis. For FACS analysis, cells were stained with anti-human CD14-PE  and anti-human CD11b-APC (Invitrogen) monoclonal antibodies. The data were analyzed using FlowJo software (BD Biosciences).+ cells as apoptotic cells, including the early apoptotic cells and the late apoptotic cells. Additionally, we performed terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to detect apoptotic K562 cells using the corresponding kit (Beyotime).Treated AML cells were stained with Annexin V/PI (KeyGEN) and subsequently analyzed by FACS. We defined all Annexin VRNA-seq analysis was performed by Capital Bio Technology . Before preparation of the RNA-seq libraries, total RNA (1 μg) was treated with a RiboMinus Eukaryote Kit  to remove rRNA. Strand-specific RNA-seq libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina  following the manufacturer’s instructions. The libraries were quality controlled with a 2100 Bioanalyzer chip  and sequenced on the HiSeq 2000 platform  with 100 bp paired-end reads.http://kobas.cbi.pku.edu.cn/index.php) for pathway analysis of the gene expression profile data for AML cells transfected with siRNA-circRNF220. Finally, Gene Set Enrichment Analysis (GSEA) v.2.0 was used to analyze a preranked list based on the DESeq2 t-statistic using preassembled gene sets from MSigDB v5.2.RNA sequencing reads were mapped to the GRCh38.p7 assembly using STAR aligner version 2.5.2b with Ensembl annotations. Data analysis was performed with R software with a low-stringency filtering scheme of a 1.5-fold change in the expression level after MAS5 normalization of all datasets. To investigate the global effect of circRNF220 on AML cells, we used KOBAS 3.0  was determined to analyze correlations. Receiver operating characteristic (ROC) analysis was applied to evaluate the power of circRNF220 expression as a prognostic marker in AML, and the value of (CtcircRNF220-CtGAPDH) served as the estimated optimal diagnostic threshold. The area under the curve (AUC), sensitivity, and specificity were also calculated. The Kaplan–Meier method was used to analyze relapse-free survival (RFS), and the log-rank test was then performed to compare the outcomes among subgroups. Finally, a Cox proportional hazard regression model was used for univariate and multivariate analysis of prognostic factors. All calculations were performed using GraphPad Prism software and R software with a two-sided P < 0.05 considered to indicate a statistically significant difference.Differences in mRNA, circRNA, or miRNA expression between two groups were analyzed using the Mann–Whitney U test for independent unpaired samples and the Wilcoxon test for paired samples. For comparisons among more than two groups, the Kruskal–Wallis test was performed first, and the Bonferroni correction for multiple comparisons was then applied. The χTo investigate the circRNA expression profile in pediatric primary AML, a high-throughput human circRNA microarray analysis was conducted using BM samples from 5 pediatric AML patients (pooled) and 5 normal individuals (control). The scatter plot visualization showed that circRNA expression levels were variable and distinguishable among the groups Fig. A. A totaWe confirmed the microarray results by qRT-PCR using the same cohort of patients and control samples used in the microarrays  derived from the RNF220 gene flanked by long introns on the right side  compared with healthy controls Fig. A, while Next, we performed ROC curve analysis to examine the potential diagnostic value of circRNF220 expression in distinguishing individuals with AML from healthy controls and individuals with other hematological diseases. The estimated optimal diagnostic threshold was 9.525, which yielded a sensitivity of 85.25%  and a specificity of 93.98% . The AUC was 0.926  , among the 87 primary AML patients initially enrolled, 11 patients (12.6%) relapsed, and the RFS rate was 87.4%. After follow-up with these patients, we found that circRNF220 expression in the patients who relapsed (n = 11) was nearly 2.5-fold higher at the time of diagnosis than that in the remaining 76 patients with continuous CR  than patients with lower circRNF220 expression in univariate analysis. Furthermore, patients with higher circRNF220 expression had a 5.866-fold higher risk of relapse  in multivariate Cox regression analysis  than patients with low circRNF220 expression, who had an RFS rate of 95.45% Fig. H. Next, Because circRNF220 was consistently upregulated in patients with primary and relapsed pediatric AML, we wondered whether circRNF220 alone is sufficient to induce leukemia progression. To evaluate the biological basis of circRNF220 dependency, we generated a vector for efficient expression of circRNF220 in AML cell lines  analysis on the commonly dysregulated transcripts. We observed that genes involved in apoptosis, lysosome and acute myeloid leukemia were positively enriched Fig. C, consis.34 Fig. D. We ind.34 Fig. E and fou.34 Fig. F. Striki.34 Fig. G. Then w.34 Fig. C. We ver.34 Fig. H-I.A numAs circRNAs often function by interacting with miRNAs, we searched for candidate miRNAs interacting with circRNF220. Through computational prediction, we identified five candidates (miR-30a/b/c/d/e). Their predicted binding sites were located within highly conserved AGO-binding sites in circRNF220 Fig. A. Using We next applied GSEA to identify pathways differentially regulated by circRNF220 inhibition and found significant downregulation of miR-30 targets, identical to the above findings Fig. G. To undTumor-specific gene expression patterns have been widely researched for their potential roles in cancer diagnosis and prognosis –43. Rela. found that circ_0009910 is significantly upregulated in AML BM samples and might be a novel prognostic biomarker in AML [CircRNAs constitute a novel class of exceptionally stable RNA species in eukaryotes. Some are abundant and evolutionarily conserved and are anticipated to be used as biomarkers in diseases , 47. Forr in AML . A new sr in AML . MoreoveStrikingly, these findings have crucial implications for improving the diagnostic yield of biopsies from patients whose BM sample yield or quality is inadequate for accurate histological diagnosis –52. BM aWe noted that circRNF220 can radically promote proliferation, accelerate cell cycle progression and suppress apoptosis in AML cell lines and primary pediatric AML BM cells. Gene expression analysis of primary AML BM cells treated with LV-siRNA-circRNF220 indicated a complex phenotype including genes related to the terms hematopoiesis and apoptotic process involved in development. These phenotypes were in accordance with those observed in in vitro experiments.CircRNAs are known to act as miRNA sponges –56. Guo Concurrent to miRNA sponge, regulation of RBPs is a plausible alternative role for circRNAs, and interactions of circRNAs with RBPs have been reported , 60. ThiRNF220 mRNA by variable cyclization, only circRNF220 was highly expressed in childhood AML. RNF220 is a RING domain E3 ubiquitin ligase that was reported to mediate the ubiquitination of multiple targets and is involved in various developmental and disease progression [RNF220 gene. We observed only a weak correlation between circRNF220 and RNF220, and it is unclear whether competition between canonical splicing and back splicing is likely to exist for the majority of loci that generate circRNF220.Given that circRNAs are resistant to RNA exonucleases, they have a sufficiently long half-life within cells . Althouggression –65. It iIn summary, circRNF220 was differentially expressed within distinct subtypes that compose the pediatric AML hierarchy, with definite prognostic value in relapse. The circRNF220-miR-30a axis operates in the progression of AML by facilitating the expression of its targets related to apoptosis. Treatment with siRNA targeting circRNF220 suppressed the growth and survival of AML cells. Collectively, these data suggest that circRNF220 should be investigated as a potentially effective biomarker and therapeutic target in pediatric AML.Additional file 1: Supplementary Fig S1. Validation the expression levels of circRNA in pediatric AML. (A) QRT-PCR results of circRNA array validation. (B) The expression levels of circRNAs identified in RNF220 gene locus. (C) The expression level of RNF220 in each patient was detected. (D) A weak positive correlation between the levels of circRNF220 and RNF220 were observed in pediatric AML specimens . (E) Comparing the expression of circRNF220 and its linear cognate gene RNF220 in pediatric AML, normalized to the expression of GAPDH and presented as the 2-△Ct value. *P < 0.05, **P < 0.01, ***P < 0.001. ns: not significant. Additional file 2: Supplementary Fig S2. ROC analysis for circRNF220 in bone marrow of AML patients. (A) The AUC indistinguishing AML and normal individual was 0.9735. (B) The AUC indistinguishing AML and ALL was 0.9209. (C) The AUC indistinguishing AML and other hematologic malignancies was 0.9274. (D)Spearman’s coefficient scatters plot of the fold-changes of circRNF220 in the BM and PB samples . BM: bone marrow, PB: peripheral blood.Additional file 3: Supplementary Fig S3. Relative level of circRNF220 in AML cells lentiviral infected with circRNF220 shRNA or circRNF220 overexpression vector. (A) Schematic of the circRNF220 overexpression vector. (B) Efficient overexpression of circRNF220 in HL-60, THP-1 and K562 cell lines. (C) circRNF220 overexpression vector did not influence parental gene expression in HL-60, THP-1 and K562 cell lines. (D) Schematic representation of the sites of the siRNA specific to the back-splice junction of circRNF220. (E) Efficient knockdown of circRNF220 in AML-K233, AML-K262, and AML-K450 three patients’ cells. (F) Specific siRNA-circRNF220 did not influence parental gene expression in AML-K233, AML-K262, and AML-K450 three patients’ cells. (G) Efficient knockdown of circRNF220 in AML-A750, AML-943, and AML-E85 three patients’ cells. (H) Specific siRNA-circRNF220 did not influence parental gene expression in AML-A750, AML-943, and AML-E85 three patients’ cells. *P < 0.05, **P < 0.01, ***P < 0.001. ns: not significant.Additional file 4: Supplementary Fig S4. CircRNF220 regulates AML proliferation and apoptosis through targeting miR-30a. (A) Correlation between circRNF220 and miR-30b, miR-30c, miR-30d, miR-30e, respectively. (B) The miR-30 family expression levels in overexpression circRNF220 by real-time PCR in HL-60. (C) RNA pulldown using biotinylated circRNF220 and detection of miR-30a in HL-60. (D) miR-30 family expression levels in AML cells transfected with miR-30a. (E) Correlation between miR-30a and MYSM1, GLUD2, ELK3, and IER2, respectively. (F) Correlation between circRNF220 and MYSM1, GLUD2, ELK3, and IER2, respectively. (G) Cell proliferation increased upon inhibitor-miR-30a and was impaired after circRNF220 KD in HL-60 cells. (H) Apoptosis was arrested by miR-30a inhibitor and which effect was eliminated after circRNF220 KD in HL-60 cells. The numbers indicate the percentages of apoptotic cells. The data are presented as the mean values of triplicates. (I) The relative expression of circRNF220 and miR-30a in AML primary cells when transfected different small interfering RNA. (J) Apoptosis was arrested upon miR-30a inhibitor treatment, and this effect was eliminated after circRNF220 siRNA transfection in primary pediatric AML cells. The numbers indicate the percentages of apoptotic primary AML cells. The data are presented as the mean values of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001. ns: not significant.Additional file 5: Supplementary Table S1. Detailed characteristics of pediatric patients with AML (n=149).Additional file 6: Supplementary Table S2. Detailed characteristics of pediatric patients with newly diagnosed Hematologic Disorders except for AML (n=147).Additional file 7: Supplementary Table S3. Sequences of the PCR primers in this study (5’-3’).


We previously reported that store-operated Ca2+ entry (SOCE) blockers, such as RP4010, are promising antitumor drugs for esophageal cancer. As a tyrosine kinase inhibitor (TKI), afatinib received FDA approval to be used in targeted therapy for patients with EGFR mutation-positive cancers. While preclinical studies and clinical trials have shown that afatinib has benefits for esophageal cancer patients, it is not known whether a combination of afatinib and RP4010 could achieve better anticancer effects. Since TKI can alter intracellular Ca2+ dynamics through EGFR/phospholipase C-γ pathway, in this study, we evaluated the inhibitory effect of afatinib and RP4010 on intracellular Ca2+ oscillations in KYSE-150, a human esophageal squamous cell carcinoma cell line, using both experimental and mathematical simulations. Our mathematical simulation of Ca2+ oscillations could fit well with experimental data responding to afatinib or RP4010, both separately or in combination. Guided by simulation, we were able to identify a proper ratio of afatinib and RP4010 for combined treatment, and such a combination presented synergistic anticancer-effect evidence by experimental measurement of intracellular Ca2+ and cell proliferation. This intracellular Ca2+ dynamic-based mathematical simulation approach could be useful for a rapid and cost-effective evaluation of combined targeting therapy drugs.Targeting dysregulated Ca Two main types of EC are distinguished by different etiological and pathological characteristics: esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). While EAC is more prevalent in the USA, ESCC predominates among Asians and male African Americans cyto by introducing variables in the computational model, as described in “Addition of TKI and SOCE Blocker in the Computational Model”. The simulated periods of intracellular Ca2+ oscillations also presented the dose-dependent inhibitory effect of RP410 or afatinib. For example, when 0.5 or 3.0 µM RP4010 was included in the computational simulation, the periods were extended to 46.3 and 83.8 s, respectively , ranging from 0.125/0.00625 to 5.0/2.5  of RP4010 and afatinib is less than 1 . The compound was dissolved in DMSO to make up a 10 µM stock solution. Afatinib was bought from Selleckchem company  and dissolved in DMSO to make a stock solution of 10 µM. Human ESCC  and normal epithelial cell line (Het-1A) were used in this study .2 incubator and maintained in a 1:1 mixture of RPMI-1640 medium and Ham’s F12 Medium  supplemented with 5% fetal bovine serum  and 1% penicillin/streptomycin .ESCC and Het-1A cells were cultured at 37 °C in a 5% CO2+ oscillations) was measured in order to calculate the corresponding periods.KYSE-150 cells were seeded in a black 96-well plate with a clear bottom. After attachment, cells were treated with RP4010 and afatinib, combined or separately, at different concentrations  and then supplemented with proteinase inhibitor cocktail . After that, protein concentrations were quantified by using a BCA kit . Primary antibodies used in this study included anti-EGFR , anti-ERBB2 , anti-ERBB3 (1:1000), anti-ERBB4 (1:1000) and anti-β-actin . Secondary antibodies included HRP-labeled goat anti-rabbit IgG (1:5000) and anti-mouse IgG . ECL substrate reagent  was used to visualize signals on ChemiDoc .4 in each well. After attachment, the cells were treated with RP4010 and afatinib, combined or separately, at different concentrations, for 24 h. For RP4010, the concentration varied from 10 to 0.625 µM. The concentration of afatinib varied from 5 to 0.3125 µM. For the combination of RP4010 and afatinib, the ratio of RP4010 to afatinib was 2:1. After 24 h, KYSE-150 cells were incubated with medium containing 10% of 3--2,5-diphenyl-tetrazolium bromide , at 37 °C. After 4 h, formazan was dissolved in 150 µL DMSO. Absorbance was measured at 570 nm on SpectraMax i3 . The survival curve was created by Graphpad Prism 5 .Cell viability was measured by using MTT assay. KYSE-150 cells were seeded in a 96-well plate at the number of 102+ signaling pathway are as follows:c denotes the Ca2+ concentration in the cytoplasm, [Ca2+ ]cyto; Ce is the Ca2+ concentration in the ER, [Ca2+ ]ER; p is the concentration of IP3, [IP3]; and h is the rate at which Ca2+ can activate IP3Rs.The mathematical model used was a modified version of one developed by Sneyd et al., with influence from Dupont et al. ,27. The J terms represent the Ca2+ fluxes across the cell and ER membranes. IP3RJ modeled the flow of Ca2+ from the ER through the IP3R channel and depended upon both [Ca2+]cyto and [Ca2+]ER. As c decreased, the difference between c and Ce increased, which resulted in an increase for IP3RJ. The parameter fk was a scaling factor used to control the IP3R density. OP represented the open probability of IP3R and depended on the activation and inactivation rates of IP3R, which were both affected by the binding of Ca2+ and IP3. The SERCA pump moved Ca2+ from the cytoplasm into the ER and changed depending on c and Ce. As Ce decreased with respect to c, the entire term increased. In order to model Ca2+ flow into the cell, inJ combined α0, which accounted for leaks into the cytoplasm through unspecified channels, and 2+ influx. These channels open and close in response to [Ca2+]ER, Ce. As Ce increased, the denominator in 2+ stores. The opposite applied if Ce decreased, meaning that the cell needs to increase ER Ca2+ stores. A Hill equation was used to model pmJ, the flow of Ca2+ through the plasma membrane pump, with pmK being the concentration of Ca2+, where half of the binding sites at the pump were occupied, and pmV is the maximum capacity of the plasma pump. The parameter γ denoted the ratio of the volume of the cytoplasm to the volume of the ER, and the parameter δ was dimensionless, as it was a scale factor relating the fluxes through the plasma membrane and ER membrane. To model IP3 concentration, pβ represents the speed it takes p to decay to the steady state sp and p ps.ν = βThe fluxes in the above equations were given as follows:α and β:The model was further expanded upon with the introduction of the functions α modeled the rate at which [Ca2+ ] inactivating IP3Rs and β modeled the rate at which [Ca2+ ] activates IP3Rs.The function 2+ resources. TKI (afatinib) and SOCE blocker (RP4010) were incorporated into the model through D1 and D2, which represent the overall drug effect on each of the channels:A new feature in this computational model was the incorporation of the effects of one drug or of the combination of two different drugs acting on different Ca2+]cyto and was expected to reduce the frequency of the oscillations. As a TKI, afatinib inhibits EGFR tyrosine kinase activity and thus affects the steady state of IP3. It was expected that it negatively affected the ER Ca2+ release through the IP3R channel and, again, reducing [Ca2+]cyto. In order to describe the relationship between the effectiveness of the drug to the dose of drug given to the cell, we used the following equations:if were described through the Michaelis–Menten kinetics:1S and 2S represent the doses of afatinib and RP4010, respectively.As a SOCE channel inhibitor, RP4010 decreased [CaAll parameters used in the mathematical model were summarized in 2+] oscillates in ESCC cells had a period of 32.8 s. We used this value to adjust the model parameters fk, α1 and τmax, accordingly, to fit. In non-tumorous esophageal epithelial cells, which contain much less SOCE and ER Ca2+ release, we divided the values of k f by 5 and α1 by 3 to reflect such differences. The working assumption was that increasing concentrations of TKI or SOCE blockers would gradually decrease the level of intracellular Ca2+ oscillations in tumor cells until to the level in Het-1A cells. Finally, to find the coefficients ci and ki in the Michaelis–Menten equations, we matched the experimental data regarding drug doses and their corresponding periods.We fitted the parameters of the mathematical model and validated it by using experimental data for different concentrations of drugs individually or in combination with a ratio of RP4010:afatinib = 2:1. The observed experimental data showed that [CaTo carry out the numerical simulations, the MATLAB’s ode23s built-in function, based on the explicit Runge–Kutta  pair of Bogacki and Shampine , was use2+ oscillations. Cancer cells present different patterns of Ca2+ oscillations during cell-cycle progression, and we focused on the G(1)/S phase, a determining phase for cell proliferation that contains majority cells with Ca2+ oscillations. To simplify the model, we trimmed the dataset by removing any data outside 1.5 times the interquartile range. After trimming, the mean values of the period calculated from the rest of the dataset were used for the mathematical simulation, as presented in In this study, at least 50 cells in each concentration group were included to calculate the period (peak–peak duration) of CaThe combination index (CI) was calculated from the CI equation algorithms by using CompuSyn software . Dose–effect Curve and combination index (CI) versus fraction affected (fa) plots were analyzed and exported from CompuSyn .


The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.Retinal guanylate cyclases (RetGCs) promote the Ca Th. Th2+ letructure  but does of Mg2+ . Thus, Mof RetGC . By contof RetGC . Ca2+ bis 100 nM ,28, wher0–500 nM , which i 5–50 nM ,18,33) we ~ 1 mM . Thereforecovery ,13, wherrk state ,21.2+ binding to the EF-hands cause GCAP1 to constitutively activate RetGC in rod and cones. Some of these mutations  are genetically linked to retinal diseases known as cone-rod dystrophies  dimeric complex [2+ are similar in structure to the four Cys residues found in the Cys4 zinc finger motif that binds to Zn2+ [4 zinc finger suggests that GCAP5 may also bind to Zn2+ in place of Fe2+. High levels of Zn2+ are found in retinal photoreceptor cells, and Zn2+ may play a role in phototransduction [2+ can bind to GCAP5 and test whether Zn2+ binding to GCAP5 can regulate RetGCs in zebrafish photoreceptors.A structural model of Feapproach   that are clustered on the surface of the protein and form a potential hot spot for drug targeting A. These


Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D)  and DHPR(E1014K) . Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.Skeletal muscle excitation-contraction (EC) coupling roots in Ca In vertebrate skeletal muscle, voltage-dependent conformational change of the skeletal muscle DHPR is transmitted via protein-protein interaction to the Ca2+ release channel - ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR), inducing its rapid opening. The resulting massive increase in cytosolic Ca2+ concentration leads to skeletal muscle contraction (Excitation-contraction (EC) coupling in skeletal muscle does not require Catury ago . Contrarcoupling , Ca2+ intraction .ncDHPR mouse  pore mutation in the EK mouse  pore mutation displayed no differences compared to wild-type (wt) mice across a broad range of tests (2+ conductance through the DHPR isoform specific for the fast (glycolytic/white) skeletal muscle  has so far not been investigated thoroughly.Recently, two independently generated genetic mouse models, the EK mouse  and the PR mouse  revisiteEK mouse , besideshenotype . Conversof tests . This N→l muscle . Since bHPR mice  are appaa+ or K+ . Neverth2+ impermeant even under conditions known to augment L-type Ca2+ currents. Our results explicitly show that the DHPR pore mutation N617D leads to an increase in Ca2+ pore binding affinity from ~1 µM (characteristic for wt DHPR) to nM range. This more than fourfold enhanced Ca2+ binding affinity is sufficient not only to completely block Ca2+ conductance through the mutant DHPR(N617D) but also does not allow permeation of monovalent cations like Cs+, Li+, and Na+ under physiological Ca2+ concentrations. This pore blocking mechanism due to atypical high-affinity Ca2+ binding in mutant DHPR(N617D) strongly contrasts the pore blocking mechanism by low-affinity Ca2+ binding in pore mutant DHPR(E1014K). As known from previous studies  locus in the outer DHPR pore region  remains Ca studies  any amine region  and the e region . With thentified  addition2+ permeation also under current enhancing conditions, we implemented corresponding experimental protocols and measured whole-cell Ca2+ currents from wt and ncDHPR myotubes isolated from new born up to 4-day-old mouse pups. As a first step, inward Ca2+ currents were recorded in the presence of 10 µM 1,4-dihydropyridine (DHP) agonist (±)Bay K 8644 applied via the standard bath solution . For voltage-gated L-type Ca2+ channels (CaV), Bay K 8644 acts as a channel opener by occupying a fenestration site at the interface of repeats III and IV in the pore region  through the wt DHPR (center and bottom), no inward Ca2+ currents (p<0.001)  or tail currents were evoked in ncDHPR myotubes under (±)Bay K 8644 administration (top and bottom).To investigate whether DHPR pore mutation N617D obstructs Cae region . Althoug2+ channels show a shift in the mode of gating not only by DHP agonist action  and long mean open times (2+ currents as well as tail currents with slower rate of current decay. To investigate whether strong depolarizations with simultaneous administration of (±)Bay K 8644 enable the entry of mutant DHPR(N617D) into mode 2 and elicit L-type Ca2+ currents, we used the pulse protocol depicted in bottom)  as well as tail current at −20 mV  when preceded by a pulse of +90 mV compared to the corresponding currents recorded without the pre-conditioning depolarization of +90 mV  (center). Conversely, no inward currents or tail currents could be evoked in ncDHPR myotubes with  or without the +90 mV pre-conditioning pulse  (top). The slight outward component at +90 mV is typically observed at strong depolarizing potentials as described previously  . Briefly; n = 5) , center. n = 10) , top. Theviously .2+ channels into mode 2 state (bottom) in the presence of (±)Bay K 8644 elicited robust, slowly inactivating L-type Ca2+ currents in wt control myotubes  (center), no inward Ca2+ currents were evoked in ncDHPR myotubes  (top), suggesting that DHPR(N617D) remained Ca2+ impermeant even under potentiating conditions. We found slight outward currents that were similar to previously observed currents in DHPRα1S-null (dysgenic) myotubes  , center,; n = 5) , top, sumyotubes  recorded2+ currents through the wt DHPR were unable to evoke Ca2+ currents through the mutant DHPR(N617D) in the ncDHPR mouse model. Out of the three, so far described mutant mammalian DHPR Ca2+ channels with ablated Ca2+ conducting ability under standard recording conditions, namely R174W (2+ currents together with enhanced tail currents in response to strong or prolonged depolarizations in the presence of (±)Bay K 8644  (top and bottom and bottom).Since both pore mutants, DHPR(N617D) as well as DHPR(E1014K) strictly prevent Ca EK mice  can evidse model  or in hese model  already channels , massive(E1014K)  but not (E1014K)  were obs+ leakiness of DHPR(E1014K) and tightness of DHPR(N617D) is also factual for other monovalent cations like the physiologically relevant Na+ ions. To clarify this conundrum, we performed patch-clamp recordings on ncDHPR myotubes under comparable experimental conditions like previously used on DHPR(E1014K) expressed in dysgenic myotubes , at near physiological (150 mM) external Na+ as the sole monovalent cation and 10 mM external Ca2+, no slow inward Na+ currents resembling the L-type Ca2+ currents were observed in ncDHPR myotubes (n = 8). Even upon reducing external Ca2+ from 10 to 1 mM to lower the blocking effect by Ca2+, DHPR(N617D) remained fully impermeant to Na+ ions . Both these observations were in stark contrast to the robust slow-activating, non-inactivating inward Na+ currents recorded from DHPR(E1014K) under comparable conditions (+ channel (NaV) isoforms (+ channel blocker tetrodotoxin (+ (top and center) can certainly be ruled out to be Cs+ currents through DHPR(N617D) since they are blocked by the K+ channel blocker TEA+  and show kinetics very different from Cs+ currents recorded from DHPR(E1014K)  and this together with TEA+ sensitivity strongly points to residual K+ currents through the endogenous delayed rectifier K+ channel (KV)  . Moreovenel (KV) . Despite2+ antagonist nifedipine. As depicted in 2+ and 150 mM Na+, exhibited nifedipine-insensitive slow outward currents (left) and rapidly activating and inactivating inward currents (right). Current-voltage relationship of outward currents  as well as of inward currents  were unaffected (p>0.05) by the presence of nifedipine. These results unambiguously confirm that DHPR(N617D) is not accountable for the outward and inward currents observed in the presence of near physiological external Na+.To directly test for a putative contribution of DHPR(N617D) in mediating the outward and inward currents described above , we meascurrents , left ancurrents , right. 2+, DHPR(E1014K) additionally lost its ion-selectivity and robustly conducts monovalent anions like Cs+ as well as physiologically relevant Na+ and K+ even in the presence of physiological concentrations of external Ca2+. Although the channel properties of DHPR(E1014K), with its charge conversion of selectivity filter glutamate E1014, are accurately explained by a widely accepted model of cardiac Ca2+ channel selectivity and permeation , inward Li+ currents were indistinguishable (p>0.05) between ncDHPR  and wt  myotubes (2+ concentration to 1 µM showed a highly significant (p<0.001) reduction of Li+ currents through ncDHPR  compared to wt  myotubes (2+ block of Li+ currents due to enhanced Ca2+ binding affinity to the DHPR(N617D) pore. In particular, at 3 µM external Ca2+, no Li+ currents could be evoked from ncDHPR myotubes  but small, significant (p<0.001) currents through the wt DHPR  were still existent , we recorded Li+ currents in ncDHPR myotubes in the presence of the 1,4-DHP antagonist nifedipine. As depicted in 2+ exhibited a drastic reduction (p<0.001) of slow inward Li+ currents . These results confirm that the slow inward Li+ currents observed in the absence of external Ca2+ are mediated by DHPR(N617D).To directly validate if the slow inward LincDHPR myotubes within the first ~20 ms of the onset of test potentials are Li+ currents through endogenous skeletal muscle Na+ channels, NaV . As demonstrated in 2+ block of inward Li+ currents through the skeletal muscle wt DHPR displays an IC50 of 1.57 µM (95% CI: 1.36–1.80 µM), which is highly comparable to published values of cardiac DHPR Ca2+ pore binding affinity (50 of 372.8 nM (95% CI: 334.4–415.8 nM) which is indeed 4.2-fold shifted to lower Ca2+ concentrations. Thus, in the mutant DHPR(N617D), introduction of the negatively charged residue D617 into the DHPR pore in close vicinity of the selectivity filter EEEE results in a significant (p<0.01) decrease in IC50 from µM to nM concentrations. Notably, also the Hill coefficient (nH) was significantly (p<0.01) different between wt DHPR  and DHPR(N617D)  (see Discussion). Since atypical high-affinity binding of Ca2+ to the mutant pore is apparently incompatible with Ca2+ conductance, this supports the idea of a mechanism by which the mutant DHPR(N617D) pore is occluded.Eventually, to quantify the impact of the DHPR pore mutation N617D on Caaffinity . Interes+ channel. Indeed, this difference in intramuscular Na+ conductance could be one of the reasons for the different phenotypes observed with the two non-Ca2+ conducting DHPR pore-mutant mouse strains, ncDHPR and EK. However, of higher physiological relevance than this Na+ conductance of DHPR(E1014K), is probably its additional massive 1,4-DHP-sensitive, non-inactivating outward K+ conductance, which again is completely absent in the DHPR(N617D) counterpart -tubular lumen may exacerbate muscle fatigability during periods of enhanced, repetitive activity  as the basis for the biophysical differences between mutant strains ncDHPR and EK, we have to take into consideration the fact that these data were derived from heterologous expression studies in tsA-201cells and not from isolated EK skeletal muscle fibers. Moreover, considering the short duration (~5 ms) of the skeletal muscle action potential (AP) (+ currents (+ flux through DHPR(E1014K) during an AP or series of APs has important phenotypic implications or not.Even though the results and interpretations of ial (AP) , slow DHial (AP) , and relcurrents , further+ and K+ leakiness of DHPR(E1014K) on the EK phenotype, 2+ permeation and/or high-affinity Ca2+ binding to the DHPR is conformationally coupled to the activation of Ca2+ / calmodulin-dependent protein kinase type II (CaMKII) and SR store refilling during sustained muscle activity. Consequently, lack of high-affinity Ca2+ binding to the DHPR(E1014K) pore causes a decrease in these Ca2+-dependent enzyme activities, ensuing alterations in the downstream Ras/Erk/mTORC1 signaling pathways and as a result decreased muscle protein synthesis and the described muscle physiological aberrations ) for accurate CaMKII activation and thus, intact downstream signaling.Besides the putative influence of the Narrations . AlthougHPR mice  exclude 2+ ions?Integration of our recent and previous findings  helped u2+ channel selectivity and permeation based on two elegant studies from the Tsien lab  of the DHPR selectivity filter. This tight embracement of Ca2+ in the DHPR pore is a prerequisite for the high selectivity for Ca2+ over Na+, K+, or other monovalent cations. However, to enable rapid passage of Ca2+ through the pore, a two-site mechanism that overcomes this tight Ca2+ binding is essential. Accordingly, the EEEE locus has been suggested to be physically flexible. Hence, irrespective that all four selectivity filter glutamates are needed to hold a single Ca2+ ion with high affinity (KD ~1 µM), their conformation can rapidly rearrange to accommodate a pair of Ca2+ ions within the pore, but then both bound with much lower affinity (apparent KD ~14 mM). This intermediate short-lived low-affinity state, together with a Ca2+- Ca2+ repulsion mechanism occurring in this doubly occupied pore, whereby one of the occupying Ca2+ ions is pushed out to the cytosolic side, is the basis for fast Ca2+ ion passage through the pore.In an attempt to answer the above questions, we intend to expand a widely accepted molecular model of Casien lab , and com2+ selectivity filter in form of the conserved EEEE locus within the pore of high threshold voltage-gated Ca2+ channels (HVA VGCC) satisfactorily explains divalent/monovalent ion selection, it neither explains the differences in the selectivity for Ca2+ among other divalent ions nor the observed distinct conductances through the different HVA VGCC isoforms  Ca2+ pore binding affinity as demonstrated by Ca2+ block of Li+ current experiments   pore  pore. Consequently, low-affinity pore-bound Ca2+ is unable to block the flux of any cation in both directions and hence Ca2+ selectivity is abolished in the mutant DHPR(E1014K). In addition, since the EEEE locus is mutated to EEKE, attraction of a second Ca2+ and subsequent competition for binding valences with the Ca2+ ion that is already bound with low affinity to this EEKE locus is impossible. Absence of this intermediate doubly occupied pore and thus, of the Ca2+- Ca2+ repulsion mechanism as the basis for fast, unidirectional Ca2+ ion passage through the pore is sufficient to explain the lack of Ca2+ conductance through the mutant DHPR(E1014K).As discussed above, proper Caeriments . Specifi of ILi+ . Althoug(E1014K) , which w4K) pore . Similaras 10 mM  again in2+ conductance through wt DHPR. The carboxyl oxygens of the DCS locus point toward the pore lumen, allowing coordination of incoming divalent cations with a preference for Ca2+ (2+ ions from the t-tubular (extracellular) side are attracted to the negative charges of the DCS locus, which in mouse DHPRα1S is formed by D296 of repeat I, E1327 of repeat IV, and supported by D615 of repeat II. This loosely bound Ca2+ ion is easily mobilized (probably by charge repulsion from excess Ca2+ ions in the t-tubule) and migrates deeper into the pore to compete with the tightly bound Ca2+ ion for binding valences of the EEEE locus . Henceforth, due to the reduced binding (µM to mM affinity), Ca2+- Ca2+ repulsion . Furthermore, metadynamics simulations show that as a consequence of this Ca2+ - Ca2+ repulsion occurring in the doubly occupied EEEE locus, one of the two Ca2+ ions moves toward the cytosolic side , is reflected in the significantly (p<0.001) lower free energy barrier  by coalescing the models discussed above? for Ca2+ . Accordifor Ca2+ , Ca2+ ioepulsion  takes pln 2+ ion . Howeverw as 6 Å , left. Fde left; . The wea barrier .2+- conducting mechanism of wt DHPR, the additional negative charge D617 in mutant DHPR(N617D), introduced in the close vicinity to the residue D615 in repeat II  pore. Congruently, molecular dynamics simulations show that immediately after the equilibration step, one Ca2+ ion is stabilized at the EEEE locus while the other Ca2+ is bound to the DCS locus  pore. Thus, simulations of pulling of Ca2+ ions through the selectivity filter of mutant DHPR(N617D) result in a significantly (p<0.001), ~8 times higher energy barrier compared to wt DHPR (2+ ions (nH) derived from four parameter logistic fit of dose-response curve is best portrayed as an ‘interaction’ coefficient, reflecting the extent of cooperativity among multiple binding sites (2+ interactions in the wt DHPR pore with its successive short-lived intermediate high and low binding affinities and repulsion mechanisms are consequently apparent in the higher nH compared to DHPR(N617D).Contrary to this smooth Caepeat II , createss right; . This tr wt DHPR , which i(2+ ions . This ra(2+ ions . The Hilng sites . The conncDHPR (2+ non-conductivity in zebrafish fast (glycolytic/white) skeletal muscle (2+ conducting DHPR of sterlet (Acipenser ruthenus), which is phylogenetically somewhere in between mouse and zebrafish, we showed , then appropriate Ca2+ pore-affinity essential for proper Ca2+ selectivity and Ca2+ conductance must exist in a surprisingly small range. Incorporation of our present and previously published data (50 for N617D) and 3.2 µM (2-fold IC50 for wt). The hampered Ca2+ selectivity and conductance mechanism of mutant DHPR(E292Q)  is blocked. Likewise, Ca2+ block of the bidirectional flux of monovalent cations, and hence Ca2+ selectivity is abolished.Interestingly, Ts3R headed into the evolution of a second DHPR isoform in zebrafish slow (oxidative/red) skeletal muscle that is likewise Canducting . This sl1S clone  yielded currents  with a sto+30 mV . As descto+30 mV , the EQ hed data  indicateR(E292Q)  is expecEE locus  cannot s2+ non-conducting DHPR was identified in the fast skeletal muscle of teleost fishes (cypriniformes) mutation N→D  is the archetypical mutation to block DHPR Ca2+ influx, in phylogenetically higher developed teleost species starting with the order lophiiformes (anglerfishes), this negatively charged D was lost by mutating to a neutral T  and highly homologous in all mammalian L-type Ca2+ channels, to positively charged K (D→K). As demonstrated previously (296 with K in a GFP-tagged rabbit DHPRα1S clone yielded mutant DHPR(D296K). Upon heterologous expression in dysgenic myotubes, this single charge conversion was sufficient to abolish inward Ca2+ currents. According to our combined model of Ca2+ selectivity and conductance and illustrated in 296 does not permit formation of an active DCS locus, and thus Ca2+ from the t-tubular (extracellular) space is no more attracted to the DCS locus. Resultantly, there is lack of easy to mobilize low-affine DCS-bound Ca2+ that would compete with the tightly EEEE-bound Ca2+ for the binding valences of the EEEE locus DHPR knock-in mouse strain, carrying a point mutation in the Cacna1s gene coding for N617D in pore loop II was described previously  or wild-type channel were enzymatically isolated and cultured in a humidified 37°C incubator with 5% CO2 as described previously . The standard bath solution for recording Ca2+ currents contained (in mM): 10 CaCl2, 145 TEA-Cl and 10 HEPES (pH 7.4 with TEA-OH). Myosin-II blocker BTS  was constantly present in the bath solution.Ionic currents were evoked by a standard 200 ms voltage-step protocol from −50 to +80 mV in 10 mV increments from a holding potential of −80 mV , unless o −50 mV . Borosil2+ channels could evoke currents through DHPR(N617D), strong or long depolarizations in the presence of racemic 1,4-dihydropyridine (DHP) agonist (±)Bay K 8644 (10 µM) were performed  to achieve near physiological Na+ concentration (150 mM). Furthermore, to test if these Na+ currents were also subject to block by Ca2+, 10 mM Ca2+ was reduced to near physiological 1 mM Ca2+.To investigate if DHPR(N617D) conducts slow-activating, non-inactivation inward Na2+ pore-binding affinity, dose-inhibition experiments for Ca2+ block of inward Li+ currents were performed. The bath solution for recording Li+ currents contained (in mM): 100 LiCl, 10 HEPES, 10 EGTA, and 25 for CaCl2 plus TEA-Cl (pH 7.4 with TEA-OH). Desired free Ca2+ concentrations (0 to 30 µM) were obtained by calibrating CaCl2 and TEA-Cl concentrations calculated using the MaxChelator simulation program (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/)  in ncDHPR myotubes, 10 µM of the 1,4-DHP antagonist nifedipine was added to the respective bath solutions.To test if the inward LiAll recordings were performed at room temperature using the Axopatch 200B amplifier , filtered at 1 kHz and sampled at 5 kHz.n = number of myotubes. Statistical significance was calculated using unpaired Student’s t-test, unless otherwise stated and was set as follows: *p<0.05, **p<0.01, and ***p<0.001.Data were analyzed and plotted using ClampFit , SigmaPlot  and Prism 8 . Data are represented as mean ± SEM and 1S - verapamil complex with a dilated intracellular gate associated to the binding of the phenylalkylamine Ca2+ antagonist drug verapamil (PDB accession number 6JPA) . For simulations, we removed the voltage-sensing domains and truncated the S5 and S6 helices of each repeat, keeping the last nine residues of the S5 and S6 helices. The C- and N-termini of each repeat were capped with acetylamide (ACE) and N-methylamide to avoid perturbations by free charged functional groups. The starting structures for simulations were prepared in MOE using the Protonate3D tool  . The stre3D tool . To neute3D tool . Using te3D tool , crystale3D tool . We addeere used . The strere used .2+ ions. Bonds involving hydrogen atoms were restrained by applying the SHAKE algorithm (For both wt DHPR and mutant DHPR(N617D), 10 ns of molecular dynamics (MD) simulations were performed in an isothermal - isobaric (NpT) ensemble using the GPU MD simulation engine pmemd.cuda  to furthlgorithm , allowinlgorithm . The Lanlgorithm  was used2+ ions along the selectivity-filter conducting pathway and their passing through the EEEE motif. As CV, we chose the distance between the center of masses (COM) of the EEEE motif residues and the upper Ca2+ ion.Metadynamics is a powerful method to explore the properties of multidimensional free energy landscapes and to enhance the sampling of configurational space in reasonable computing time . MetadynSimulations were performed at 300 K in an NpT ensemble. We used a Gaussian height of 1.5 kJ/mol and width of 0.1 nm. For both wt DHPR and mutant DHPR(N617D), five repetitions of metadynamics runs, each 10 ns, were performed. Pymol Molecular Graphics System was used to visualize the key interactions and differences between wt DHPR and mutant DHPR(N617D) pore conductances. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ conductance of the zebrafish skeletal muscle dihydropyridine receptor (DHPR). The authors show that introduction of the residue responsible for eliminating Ca2+ conduction of the zebrafish DHPR (N617D) into the mouse skeletal muscle DHPR abolishes Ca2+ and monovalent cation conduction by causing a 4.2-fold increase in high affinity Ca2+ binding and block of the channel pore. This mechanism of inhibition of Ca2+ permeation is distinct from that achieved by a mutation (E1014K) within the selectivity filter, in which the channel is impermeable to Ca2+ but still conducts monovalent cations. The results provide insight into both how ion conduction is eliminated in the DHPR of teleost fish and the surprisingly different phenotypes of two knock-in mouse models (N617D and E1014K) that abolish DHPR Ca2+ influx based on these two distinct mechanisms.This study provides convincing experimental evidence for the biophysical mechanism responsible for lack of CaDecision letter after peer review:2+ influx due to atypical high-affinity Ca2+ binding" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Henry M Colecraft as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Stephen Cannon (Reviewer #2).Thank you for submitting your article "Pore mutation N617D in the skeletal muscle DHPR blocks CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:2+ currents even under conditions known to augment Ca2+ conduction of WT DHPR channels  and also do not exhibit detectable inward Na+ conductance in the presence of a physiological Na+ concentration even when extracellular Ca2+ is reduced to 1 mM. The mechanism of inhibition of Ca2+ permeation is distinct from that achieved by mutation (E1014K) within the selectivity filter. The results provide insight into both how ion conduction is eliminated in the DHPR of teleost fish and the surprisingly different phenotypes of two knock-in mouse models (N617D and E1014K) that abolish DHPR Ca2+ influx based on these two distinct mechanisms.The manuscript identifies a novel mechanism for abolishing Ca permeation through voltage-dependent Ca (CaV) channels. The authors show that the N617D mutation in CaV1.1 introduces a second high-affinity Ca binding site (Kd ~0.4 uM) at a divalent cation selectivity (DCS) that is separate from the high affinity site (EEEE) within selectivity filter (kd ~1.6 uM), and that the two sites do not exhibit significant electrostatic repulsion when Ca is bound. The results show that skeletal myotubes from N617D knock-in mice do not exhibit detectable inward CaEssential revisions:Two main areas were identified as points needed to be addressed by the authors to strengthen the conclusions of the work.2+ in Figure 3 are mediated by N617D channels should be directly verified with the use of a DHP antagonist.1. The conclusions that the inward/outward currents observed in Figure 2 are not mediated by N617D channels, while the inward Li+ currents in the absence of Ca2. The final section of the paper presents a conceptual model for how the apparent increase in Ca binding affinity severely reduces Ca flux (i.e. "block") and at the same time Ca occupancy of the EEEE motif preserves selectivity over monovalent cations. The key concept is that N617D enhances the Ca binding at the DCS site (0.4 μm affinity) which lies in the pore vestibule external to EEEE. It is proposed that this high-affinity binding at DCS impedes Ca transition to the EEEE site. A potential problem with this model is that if the DCS → EEEE translocation of Ca is too slow to allow "double occupancy" of EEEE by Ca (and thereby reduce EEEE affinity to mM range and promote flux) then how does the EEEE site get occupied with single Ca? The on-rate for initial block of Li current when adding Ca would be very slow. Quantitative simulation would be very helpful to demonstrate it is possible for the DCS → EEEE translocation of Ca to be slow enough to prevent a detectable Ca conductance, and yet at the same time be sufficiently fast to establish Ca block of Li current in a reasonable amount of time. Essential revisions:Two main areas were identified as points needed to be addressed by the authors to strengthen the conclusions of the work.2+ in Figure 3 are mediated by N617D channels should be directly verified with the use of a DHP antagonist.1. The conclusions that the inward/outward currents observed in Figure 2 are not mediated by N617D channels, while the inward Li+ currents in the absence of Ca+ currents in the absence of Ca2+ in Figure 3 are mediated by DHPR(N617D). Both our claims could be verified by these experiments and according text blocks have been added to the Results  and Material and Methods (lines 490-493) sections. Additionally, a new figure panel  and a new figure  with the corresponding legends have been included.In agreement with the reviewers’ request, we performed additional patch-clamp experiments in the presence of 10 µM of the 1,4-DHP antagonist nifedipine in the external recording solution to directly verify that the inward / outward currents in Figure 2 are not mediated by DHPR(N617D) channels, whereas the inward Li2. The final section of the paper presents a conceptual model for how the apparent increase in Ca binding affinity severely reduces Ca flux (i.e. "block") and at the same time Ca occupancy of the EEEE motif preserves selectivity over monovalent cations. The key concept is that N617D enhances the Ca binding at the DCS site (0.4 μm affinity) which lies in the pore vestibule external to EEEE. It is proposed that this high-affinity binding at DCS impedes Ca transition to the EEEE site. A potential problem with this model is that if the DCS → EEEE translocation of Ca is too slow to allow "double occupancy" of EEEE by Ca (and thereby reduce EEEE affinity to mM range and promote flux) then how does the EEEE site get occupied with single Ca? The on-rate for initial block of Li current when adding Ca would be very slow. Quantitative simulation would be very helpful to demonstrate it is possible for the DCS → EEEE translocation of Ca to be slow enough to prevent a detectable Ca conductance, and yet at the same time be sufficiently fast to establish Ca block of Li current in a reasonable amount of time.2+ ions passing through the selectivity filters of wt DHPR and mutant DHPR(N617D). We found that in wt DHPR immediately after the equilibration phase, two Ca2+ ions compete for the EEEE locus. Consequently, Ca2+ – Ca2+ repulsion occurs in this doubly occupied EEEE locus, so that the first of the two Ca2+ ions is pushed towards the cytosolic side. Moreover, metadynamics simulations capturing the passage of Ca2+ ions through the pore revealed a significantly smaller energy barrier for the wt DHPR selectivity filter compared to mutant DHPR(N617D).In order to address these queries, we performed molecular dynamics (MD) simulations in combination with an enhanced sampling technique  to estimate the differences in free energy profiles of Ca2+ ion stabilized in the EEEE locus and the other bound to the DCS locus. This shows that already in a very short equilibration phase, the Ca2+ ions reach and occupy the EEEE and DCS loci, and since ions cannot pass though the selectivity filter regions anymore, this result in complete occlusion of the pore. This additional stabilization at the DCS locus that a Ca2+ ion experiences while moving towards the cytosolic side is also reflected in the ~ 8-times higher energy barrier compared to wt DHPR. The slow detachment and thus slow migration of the Ca2+ ions towards the cytosolic side, as observed in our simulation study, suggests occlusion of the pore without the force applied during simulations to pull the Ca2+ ions through the pore.MD simulations on mutant DHPR(N617D) revealed a different picture. Already 1 ns after the equilibration phase, we find one CaObservations from the simulation experiments are presented in a new figure  and the Discussion section .2+ to the EEEE locus results in complete occlusion of the DHPR(N617D) pore, even for monovalent cations, as demonstrated above”  from the Discussion section. Misleadingly it suggested that divalent and monovalent cations can only be blocked at the continuously Ca2+- occupied EEEE locus in mutant DHPR(N617D), a statement for which no experimental evidence exists. Considering the tight dimension of the pore around the DCS locus, it is very likely that cations are blocked already at the Ca2+- occupied DCS locus. This mechanism would allow a very fast on-rate for the initial block of Li+ currents when adding Ca2+ ions to the external solution, independent of the speed of the DCS → EEEE translocation, which can be slow enough to prevent detectable Ca2+ conductance. Detailed, quantitative MD simulations with reliable time scales and interaction energies to predict if the DHPR(N617D) channel pore can be blocked via the Ca2+- occupied DCS locus is a challenging aim, but considering the massive need of computing resources would require an independent study.As a consequence of these new insights, we removed the sentence “Additionally, undisturbed continuous high affinity binding of Ca