SLC10A7 gene are associated with skeletal dysplasia and amelogenesis imperfecta and reveal loss of function on cellular calcium influx. More recently, an additional disease-related genetic variant (P303L) as well as some novel genetic variants  have been identified. In the present study, these variants were expressed in HEK293 cells to study their subcellular localization and their effect on cellular calcium influx. All variants were properly sorted to the ER compartment and closely co-localized with the STIM protein, a functional component of SOCE. The variants P303L and L210F showed significantly reduced effects on cellular calcium influx compared to the wild type but still maintained some degree of residual activity. This might explain the milder phenotype of patients bearing the P303L variant and might indicate disease potential for the newly identified L210F variant. In contrast, all other variants behaved like the wild type. In conclusion, the occurrence of variants in the SLC10A7 gene should be considered in patients with skeletal dysplasia and amelogenesis imperfecta. In addition to the already established variants, the present study identifies another potential disease-related SLC10A7/RCAS variant, namely, L210F, which seems to be most frequent in South Asian populations.The solute carrier family 10 member SLC10A7 is a negative regulator of intracellular calcium signaling (RCAS). In cell culture, SLC10A7 expression is negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. SLC10A7-deficient cells have significantly increased calcium influx after treatment with thapsigargin for depletion of ER calcium stores, whereas SLC10A7/RCAS overexpression limits calcium influx. Genetic variants in the human  The concentration of free Ca2+ ions is much higher in the extracellular than in the intracellular compartment. In addition, Ca2+ is sequestered in the endoplasmic and sarcoplasmic reticulum (ER/SR), from where it can be released for rapid cellular signaling (2+ to enter cells via the plasma membrane (2+ binding domain (EF hand) in the luminal side of the ER, represents a Ca2+ sensor  is terminated  belongs to the solute carrier family 10 of bile acid and steroid sulfate membrane transporters (2+ influx after treatment with thapsigargin (TG), ionomycin, and ATP/carbachol treatment, which are commonly used for depletion of ER Ca2+ stores. Furthermore, SLC10A7-deficient cells showed significantly higher intracellular Ca2+ levels. In contrast, SLC10A7/RCAS overexpression significantly reduced the Ca2+ influx, clearly pointing to a role of the SLC10A7/RCAS protein as negative regulator of intracellular calcium signaling. However, the exact molecular function of SLC10A7/RCAS as a transporter or regulator molecule is still not clear. However, there are several hypotheses about its function. (I) SLC10A7 might limit the transport capacity of SERCA or might increase the rate of Ca2+ leaking from the ER. (II) SLC10A7 might negatively regulate STIM and/or ORAI, e.g., by affecting the sensitivity of STIM to Ca2+, or by decreasing the probability of ORAI opening. (III) SLC10A7/RCAS might also play a role for STIM-ORAI complex formation or the stability of this complex at the plasma membrane .Unless otherwise stated, all chemicals, including TG (T9033) and probenecid (P8761), were from Sigma-Aldrich . Fluo-4 AM (F14201) was purchased from Thermo Scientific . Ca2, and 95% humidity.GripTite 293 MSR cells  were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal calf serum , 1% Minimum Essential Medium of Non Essential Amino Acids (Gibco), L-glutamine , penicillin , and streptomycin  at 37°C, 5% COEscherichia coli (E. coli). Grown colonies were picked and plasmids were isolated with the GeneJET Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer’s protocol. Finally, the generated STIM-GFP was verified by DNA sequencing (Seqlab Microsynth).C-terminally mScarlet-tagged constructs for STIM1, ORAI1, and SERCA2b were generated as reported previously for SLC10A7 transcription variant v2 ”; DpnI enzyme  for 1 h at 37°C and transformed into TOP10 chemically competent E. coli. Plasmids were isolated with a GeneJET Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer’s protocol, and the generated point mutations were verified by DNA sequencing (Seqlab Microsynth).Site-directed mutagenesis was performed to create the described point mutations in SLC10A7-v2-mScarlet (in pcDNA3.1 (+) vector). Forward and reverse primers were designed and are listed in 2+ imaging, HEK293 cells (6.0 × 104 per well) were seeded into 96-well plates  coated with poly-L-lysine. Then 6 h after seeding, the cells were transiently transfected with 0.5 µg SLC10A7-mScarlet WT or mutant plasmid DNA with Lipofectamine 2000  according to the manufacturer’s protocol. After 40 h of culturing, the medium was replaced with fresh serum-free medium for 2 h. Subsequently, the cells were incubated in Ca2+-free HEPES buffer containing 2 µM Fluo-4 AM and 1 mM probenecid for 15 min at room temperature. Afterward, the cells were washed gently three times with Ca2+-free HEPES buffer and incubated for an additional 15 min to allow complete de-esterification of intracellular AM esters. Then the cells were incubated with 1 μM TG at room temperature. After 5 min of treatment with TG basal fluorescence was recorded for 1 min with a DM5500 Leica fluorescent microscope . After these 60 s, 2 mM Ca2+  was added and Ca2+-induced fluorescence was recorded every 10 s for an additional minute. The fluorescence signal was determined with LAS-X  for approximately 80 defined regions of interest of single cells. Data are presented as the mean background-subtracted fluorescence intensity of each cell normalized to the intensity of the first image (F/F0).For Ca4 per well) were seeded into 8-well µ-slides  coated with poly-L-lysine. Then 6 h after seeding, the cells were transiently transfected with 1 µg plasmid DNA (0.5 µg GFP construct and 0.5 µg mScarlet construct) with Lipofectamine 2000  according to the manufacturer’s protocol. After 40 h of culturing, the cells were washed with PBS and incubated with or without 2 μM TG for 10 min at 37°C. Subsequently, the cells were washed with PBS three times and fixed with 3% PFA  for 20 min at room temperature. Thereafter, the cells were washed and loaded with 200 µL PBS per well. Z-stack cell imaging of approximately 50 single cells per well was performed as described below. For co-localization with green fluorescent organelle markers, cells were seeded and transiently transfected as described above, except only 0.5 µg mScarlet construct (SLC10A7-/STIM1-/ORAI1-/SERCA2b-mScarlet) were used for Lipofectamine 2000 transfection. After 24 h of culturing, the cell medium was changed and approximately 30 particles per cell CellLight Reagent BacMam 2.0 (Invitrogen) containing a signal peptide fused to emerald GFP were added to allow expression in the following compartments: ER (C10590), late endosomes (C10588), lysosomes (C10596), early endosomes (C10586), Golgi (C10592), peroxisomes (C10604), and mitochondria (C10600). After 16 h of culturing, the cells were washed with PBS and fixed as described above. Then Z-stack cell imaging of approximately 5–48 single cells per well was performed. All co-localization studies were performed at room temperature on an inverted Leica DM5500 fluorescence microscope (Leica). Images were generated using a 63 × oil objective and green (488 nm) and red (568/594 nm) filter sets, respectively. The Z-stack (9.5 µm size) of single cells was recorded. Subsequently, Pearson’s correlation coefficients (PCC) were calculated with the LAS-X imaging software.For co-localization studies, HEK293 cells . Error bars represent means ± SD. The number of samples, number of experimental repetitions, and significance level are indicated in the figure legends.https://gnomad.broadinstitute.org) lists more than 650 genetic variants for the human SLC10A7 gene. From these we filtered out the 540 variants that occur in exon sequences and further selected 140 missense variants (https://sift.bii.a-star.edu.sg) and PolyPhen (https://genetics.bwh.harvard.edu) predictions, and these variants  were experimentally analyzed in the present study  and green (Fluo-4) fluorescence was recorded every 10 s for 1 min. As reported before, overexpression of the SLC10A7-mScarlet WT construct significantly limited Ca2+ influx, with a ratio of 1.7  and protein expression comparable to those of the WT SLC10A7 protein  with its major components STIM and ORAI is a central mechanism in cellular Ca2+ signaling. Several regulatory factors of the STIM/ORAI complex have been described, including the CRAC channel regulator 2A  and six novel potentially disease-related variants  were analyzed. We first examined whether these variant proteins are sorted as the WT SLC10A7/RCAS protein, and then we determined their effects on SOCE.The store-operated CaCRACR2A;  and the CRACR2A; . Recentlignaling . Express2+ influx experiments in the present study. In subsequent co-localization studies of SLC10A7-mScarlet and STIM-GFP we found a high degree of overlay, which indicates that large part of the SLC10A7/RCAS protein is localized in the ER in close proximity to STIM. However, it should be mentioned that previous studies found different sorting of the SLC10A7/RCAS protein. Using different FLAG- and HA-tagged SLC10A7 constructs, Candida albicans is at least partly localized in the plasma membrane  and sulfated steroid hormones (SOAT/SLC10A6) . All othmembrane . The lat oocytes . In anot oocytes . Also, A oocytes . Based o2+ influx. We examined the co-localization of mScarlet-tagged SLC10A7/RCAS and GFP-tagged organelle markers in HEK293 cells and found the highest degree of co-localization with the ER marker. ER expression of SLC10A7-mScarlet was further verified by co-localization studies with STIM-GFP, which is typically located in the ER. However, it has to be mentioned that the degree of ER localization was slightly higher for STIM and SERCA compared to RCAS, which indicates that at least part of the SLC10A7/RCAS protein might also be sorted to the Golgi and plasma membrane. As a dynamic sorting regulation is known for STIM, all sorting studies were additionally performed after ER Ca2+ depletion by TG treatment. As expected, after treatment with TG STIM lost some of its co-localization with SERCA but increased its co-localization with ORAI, which reflects quite well the physiological regulation of STIM sorting under ER Ca2+ depletion  and therefore are an appropriate model for transient overexpression of the respective variant SLC10A7/RCAS proteins. Ca2+ flux measurements were performed in cell culture plates with transiently transfected cells, where cells with SLC10A7-mScarlet-derived red fluorescence were classified as transfected and cells without red fluorescence were classified as untransfected controls. As previously described by 2+ influx in cells pretreated with TG after the addition of 2 mM Ca2+ to the extracellular medium. It is interesting that for five of the novel SLC10A7 variants  no significant effect on Ca2+ influx could be detected compared to the WT, which indicates that contrary to the bioinformatics prediction the function of these SLC10A7/RCAS variant proteins was not affected. In contrast, the disease-related variant P303L  in a cellular system. For these experiments, we included the disease-related loss-of-function variant L74P as a control. We first investigated whether these variants are sorted identically as the WT protein and whether they show a similar degree of co-localization with STIM. It is interesting that the two disease-related variants, namely, L74P and P303L, showed a significantly higher degree of co-localization with STIM compared to the WT. This might indicate an effect of the mutation on normal sorting behavior that might also contribute to defective function of these SLC10A7/RCAS variant proteins . In contnt P303L  that hadeviously .SLC10A7 variants . Interestingly, In addition to the already established disease-related variants , the occSLC10A7 gene should be considered in patients with skeletal dysplasia and amelogenesis imperfecta. In addition to the already established variants, the present study identifies another potential disease-related SLC10A7/RCAS variant, namely, L210F, which seems to be most frequent in South Asian populations.In conclusion, the occurrence of variants in the


Aconitum, which has been proved to have severe cardiovascular toxicity. The toxic effect of aconitine on cardiomyocytes is related to intracellular calcium overload, but the mechanism remains unclear. The aim of this study was to explore the mechanism of aconitine inducing intracellular Ca2+ overload and promoting H9c2 cardiomyocyte apoptosis through transient receptor potential cation channel subfamily V member 2 (TRPV2). After treated with different concentrations of aconitine, the level of cell apoptosis, intracellular Ca2+, and expression of p-p38 MAPK and TRPV2 of H9c2 cardiomyocytes were detected. The results showed that aconitine induced Ca2+ influx and H9c2 cardiomyocyte apoptosis in a dose-dependent manner and promoted p38 MAPK activation as well as TRPV2 expression and plasma membrane (PM) metastasis. siTRPV2, tranilast, and SB202190 reversed intracellular Ca2+ overload and H9c2 cardiomyocyte apoptosis induced by aconitine. These results suggested that aconitine promoted TRPV2 expression and PM metastasis through p38 MAPK signaling, thus inducing intracellular Ca2+ overload and cardiomyocyte apoptosis. Furthermore, TRPV2 is a potential molecular target for the treatment of aconitine poisoning.Aconitine is the main effective component of traditional Chinese medicine  Aconitum species, containing aconitine, are widely used in the clinical treatment of rheumatism, arthritis, bruise, fracture, pain, and other diseases in China and other Asian countries because of their medicinal properties. However, due to their potential proarrhythmic effect, Aconitum and its related preparations are now restrictively used in the treatment of rheumatoid arthritis and some cardiovascular diseases [Aconitum, aconitine is a natural diterpene alkaloid [diseases . As the alkaloid , which walkaloid . Althougalkaloid , 5, its alkaloid . Growingalkaloid . Accordialkaloid , 9, eneralkaloid , oxidatialkaloid , inflammalkaloid , and so alkaloid . However2+ permeability channel. In myocardial tissue, TRPV2 was expressed in intercalated discs of the sarcomere and participated in the maintenance of appropriate electromechanical coupling and structure of the cardiomyocyte and myocardium [2+ influx and induces intracellular calcium overload [2+ signal [2+ overload by activating the p38 MAPK signal, leading to arrhythmia and cardiomyocyte apoptosis [2+ overload and apoptosis are related to TRPV2 expression, PM metastasis, and p38 MAPK signal activation.Transient receptor potential cation channel subfamily V member 2 (TRPV2) is a stretch-sensitive Caocardium . It is aoverload . Studiesoverload , 16. Mea+ signal , and acopoptosis . p38 MAPpoptosis . Studiespoptosis –21, whicpoptosis . However2+ influx through the p38 MAPK signal and thus promote cardiomyocyte apoptosis. The present study was designed to investigate this possibility in vivo which used the rat cardiomyocyte H9c2 cell line.According to the above, here we hypothesized that aconitine could induce TRPV2-mediated Ca2 humidified incubator. Aconitine (content ≥ 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). H9c2 cells were treated with different dosages of aconitine  for 24 h.The rat cardiomyocyte H9c2 cells  were cultured in the DMEM  with 10% FBS  and 0.1% penicillin-streptomycin solution  at 37°C in a 5% CO® 2000  according to the manufacturer's protocols. Tranilast  was used to inhibit the activation of TRPV2 at a dosage of 75 μM. SB202190  was used to inhibit the activation of p38 at a dosage of 10 μM.TRPV2 siRNA and negative siRNA were obtained from Shanghai GenePharma Co., Ltd., and transfected into H9c2 cells with a final concentration of 10 nM for 6 h by Lipofectamine5/100 µL. Subsequently, the cells were incubated for 30 minutes at room temperature in the dark with FITC annexin V and propidium iodide  and then further diluted with 400 µL 1X annexin binding buffer. Then, the fluorescence intensity of the cells was quantified by using a flow cytometer  using fluorescence emission at 530 nm (FL1) and >575 nm (FL3). Single color stains for FITC annexin and PI and unstained cells were included in all experiments as positive and negative controls.Flow cytometry assay was used to measure the cell apoptosis rate according to the previous report . The apo2+ concentration variations were detected using the fluorimetric assay with Fura-2AM (a ratiometric probe for intracellular Ca2+) as essentially described [2+ were evaluated by fluorescence (510 nm) emission ratio at excitations at 340 and 380 nm, which can directly reflect the intracellular calcium content. In brief, the H9c2 cells were suspended in the buffer without Ca2+, and the basic fluorescence level was recorded; then, Ca2+ was applied at 2 mM leading to calcium influx through opened membrane calcium channels  after 20 s. In order to detect the influence of aconitine and p38 MPAK/TRPV2 inhibitors on the intracellular calcium content, they were added into the resuspended cells just before the beginning of the measurement, then each group of H9c2 cells was resuspended in buffer solution containing 2 mM Ca2+, and their fluorescence levels were recorded. To calculate the effect of inhibitors to aconitine, the normalized Ca2+ influx in the absence of aconitine and the channel activity at 2 mM Ca2+ are used as reference points.Intracellular Caescribed . The cha−ΔΔCq method [The performance of the qRT-PCR assay followed the methods of Peng et al. with some modification . Total Rq method . The priβ-actin  was used as a loading control. Each experiment was performed in triplicate.Western blotting was used to examine the expression levels of proteins in this study; the method followed the methods of Peng et al. with some modifications . Each grP < 0.05 was considered to indicate a statistically significant difference.All the data are presented as the mean ± SEM. Differences between two or multiple groups were evaluated using a one-way ANOVA or two-way ANOVA followed by Bonferroni post hoc test. Statistical analyses were performed by GraphPad Prism 7.0 software (GraphPad Software Inc.). μM for 24 h, and then the cell apoptosis and intracellular Ca2+ were measured. As the results show, aconitine induced obvious H9c2 cell apoptosis at a lower dose of 0.25 μM, and the effect of aconitine on H9c2 cardiomyocyte apoptosis was dose dependent  were used to inhibit TRPV2 expression and function. The results showed that TL had no obvious influence on the expression level of TRPV2, both mRNA and protein, which indicated TL could not inhibit the expression of TRPV2 but can inhibit the activation of TRPV2, while siTRPV2 significantly decreased the expression of TRPV2 mRNA and protein stimulated by aconitine (1.0 μM) , which indicated that TL has no obvious influence on TRPV2 expression but inhibits the activation of TRPV2, and TRPV2 inhibition by TL and siTRPV2 could reverse the Ca2+ influx induced by aconitine. These results suggested that the Ca2+ influx induced by aconitine was partly depended on the expression and activation of TRPV2.To verify whether aconitine induced Ca Figures . ImportaμM)  was used to inhibit the activation of p38 MAPK. As the results show, SB could effectively downregulate both mRNA and protein expression of TRPV2 stimulated by aconitine . SB downregulated the expression of cleaved caspase-3, p-p38, and membrane TRPV2 induced by aconitine, while TL just reduced the expression of cleaved caspase-3 and membrane TRPV2 significantly but had no obvious influence on the expression of TRPV2 (Figures 2+ influx promoted by aconitine (2+ overload and apoptosis.To further verify whether aconitine promoted TRPV2 expression and PM translocation through the p38 MAPK signal, thus mediating intracellular Ca Figures . In addiconitine . These rAconitum, as a world-famous medicinal plant, has been used for more than 2000 years. However, aconitine, as the main effective component of Aconitum, is a diterpenoid alkaloid with severe cardiovascular toxicity, which often induced tachyarrhythmia and hypotension and led to high mortality [α/NLRP3 signal transduction [2+ overload [2+ influx in a dose-dependent manner (ortality , 28. Presduction , Notch1-sduction , mitochosduction , p38 MAPoverload . In the t manner , and thet manner .+ channel, Ca2+ channel, and K+ channel [2+ arrhythmia is most often reported to be related to the cardiotoxicity of aconitine [2+ [2+ and can be activated by lipids [2+ influx, thus accelerating disease progression [2+ overload plays an important role in maintaining cardiomyocyte activity and cardiac function [2+ influx stimulated by aconitine (2+ influx (2+ influx by activating the p38 MAPK signal. Furthermore, tranilast and SB202190 reduced the H9c2 cardiomyocyte apoptosis stimulated by aconitine (2+ overload promoted by p38 MAPK activation.Growing evidence showed that the arrhythmia induced by aconitine is related to the activity of various ion channels, such as Na channel , 32; amoconitine , 17, 33.tine [2+ . TRPV2 iy lipids . Under pgression , which igression , 36. Prefunction . The resconitine . Previou+ influx , which iconitine . These rin vivo assays in the present study, and results showed that aconitine promoted intracellular Ca2+ overload and cell apoptosis of H9c2 cardiomyocytes in a dose-dependent manner. Furthermore, aconitine promoted TRPV2 expression and PM metastasis through p38 MAPK signaling, thus inducing apoptosis mediated by intracellular calcium overload. These results indicated that TRPV2 may be a potential molecular target to treat aconitine poisoning and protect cardiac function.In summary, the effect of aconitine on cardiomyocytes was detected by


Neurotrauma assumes an instant or delayed disconnection of axons (axotomy), which affects not only neurons, but surrounding glia as well. Not only mechanically injured glia near the site of disconnection, especially transection, is subjected to the damage, but also glia that is remote from the lesion site. Glial cells, which surround the neuronal body, in turn, support neuron survival, so there is a mutual protection between neuron and glia. Calcium signaling is a central mediator of all post-axotomy events, both in neuron and glia, playing a critical role in their survival/regeneration or death/degeneration. The involvement of calcium in post-axotomy survival of the remote, mechanically intact glia is poorly studied. The purpose of this review is to sum up the calcium-involving mechanisms in responses of neurons and glial cells to axotomy to show their importance and to give some suggestions for future research of remote glia in this context. Traumatic injuries to the central nervous system  affect young and middle age people causing premature death and disability ,2. TheseThe neurons’ vulnerability to AT depends on a number of factors, such as localization, age, and distance. Neurons in the peripheral nervous system (PNS) usually survive axotomy and regenerate, while many neurons in the central nervous system (CNS) undergo degeneration and death after axotomy. This is related to neuronal factors, such as differences in gene expression in cell response to axotomy, and non-neuronal factors, such as immune proteins, which inhibit the regeneration, and the interaction of both types of factors ,4. In veTwo forms of axotomy are now considered. “Primary” axotomy occurs when axons are broken apart or transected directly in the mechanical impact on nervous tissue. The physical transection of axons massively involves its microenvironment and creates a dramatic, immediate disturbance of ionic regulation. “Secondary” axotomy occurs after relatively minor lesions to axons, such as stretch injury or diffuse axonal injury in brain trauma, which trigger a cascade of events, ultimately resulting in cytoskeleton degradation and axonal rupture . In the 2+ is critically involved in a number of signal pathways, controlling cell homeostasis. It plays an important role in neurodegeneration o elevation increases pHi and decreases [Na+]i, and vice versa oce versa .2+ ATPase (PMCA), which regulates intracellular Ca2+ concentration by taking calcium out from the cell i between neurons, axotomized 7–10 days before, and controls. However, the elevation of [Ca2+]i, caused by orthodromic and antidromic stimulation, and the recovery after the stimulation train, were significantly lower in axotomized neurons, than in controls.The level of intracellular Case to AT . In 38]2+ mediatPerineuronal glial cells are a specific cell type for the peripheral nervous system and play a critical role in the conditions of peripheral nerve damage. These cells are located in optimal fashion so that, to affect the functions of sensory neurons, for example, they envelope sensory neurons in the structure of dorsal root ganglia. The barrier they create, usually one-to-three layers, slows down the diffusion of many molecules, especially large ones, which gives a certain control of perineuronal media and helps to maintain in it the required homeostasis for the action potential propagation in the sensory neurons .2+ waves in the glial envelope as a result of mechanical damage or a signal from an axon [In response to peripheral nerve damage, a number of interconnected processes occurs in neuron and satellite glia. This includes the coupling of satellite glial cells, increase in ectopic spontaneous neuronal spiking and abrupt changing in electrical characteristics of neurons and glia  , glial p an axon .+ ones, between these cells. Additionally, the decrease in the expression of inward rectifying K+ channel Kir4.1 in satellite glial cells, associated with sensory nerve AT, also affects this pain condition. Thus, changes in the levels of expression of Cx43 and Kir4.1 can indirectly affect threshold values of action potential via changing the ability of satellite glial cells to effectively buffer the extracellular perineural medium [+, K+, Cl−, and Ca2+ channels function in Schwann cells and astrocytes [Upon the disturbance of homeostasis, as in the case of peripheral nerve injury, satellite glial cells become active, demonstrating a high expression of GFAP , and proliferate. Damage to peripheral nerves also induces an increase in the expression of the gap junction subunit connexin 43 (Cx 43) in satellite glial cells of dorsal root ganglia ,42. The l medium . Voltagetrocytes .At the ultrastructural level, after AT, glial and/or vesicular seal and growth cone for regeneration can be formed. The glial seal formation on the end of severed axon appears to be typical for crustaceans. The intrusion of glial cell was more significant in areas near to the axotomy site and increased with time. Glia, absorbed in the stumps, demonstrates hypertrophy and changes in nucleus morphology. At the same time, despite serious changes in morphology, these stumps still preserved the ability to conduct action potentials and to release neurotransmitters in their synapses .2+ occurs mainly due to its release from intracellular storages, then the more gradual and continuous dysregulation of intracellular Ca2+ metabolism takes place. There is a suggestion that physical impacts can induce “mechanoporation”, an opening of axolemma, causing intracellular Ca2+ currents. The focal elevation of Ca2+ in axons is linked to the formation of axonal spherical swellings. The formation of these spheroids can be prevented by blocking Na+–Ca2+ exchangers NCX-1, voltage-gated N- and L-type Ca2+ channels, thus probably preventing reaching the Ca2+ threshold level for axolemma. It is still questionable if the prevention of such processes beneficial, but, apparently, the non-controlled elevation of intracellular Ca2+ level can induce secondary AT. The possible mechanism of this includes active cytoskeleton degradation, mediated by Ca2+-dependent cleaving enzymes, activated by Ca2+ elevation. Furthermore, Ca2+ uptake by mitochondria can induce energetic dysfunction, forming reactive oxygen species and resulting in oxidative injury [In CNS axon stretching, an acute elevation of intracellular Cae injury .2+ in major or minor lesions urges different glial or axonal membrane vesicle structures to accumulate in the lesion site and interact, repairing the integrity of axolemma through Ca2+-induced fusions [2+ and is most likely mediated by membrane fusion proteins. Ca2+ also regulates the interaction between synaptotagmin and syntaxin, which are believed to mediate the fusion of synaptic vesicles with axolemma, providing an opportunity for neurotransmitter release in synapses. To determine if the synaptic proteins are involved in axon sealing after the damage, the researchers administrated antibodies into giant squid and crayfish axons, which inhibited either binding Ca2+ ions with Syt or the Ca2+-dependent interaction of syntaxin with Ca2+-binding domain of synaptotagmin. Axons, treated with antibodies for synaptotagmin and syntaxin, were not sealed after AT, which indicates that these proteins play a significant role in repair of neuronal membrane after AT [2+-dependent protease, promotes sealing of transected axons in squid; in crayfish, calpain inhibitors prevent the sealing [2+ transient in mechanical damage also induces growth cone formation [The influx of Ca fusions . The seaafter AT ,47. Calp sealing ,49. It i sealing  and part sealing ,52. Ca2+ormation .Periplaneta americana, the proliferation of perineural glial cells began soon after axotomy [In a number of cases, axon injury leads to the proliferation of surrounding glia, expressed both in a direct increase in glial extension and an increase in the number of glial cells via their division. In experiments with giant nerve fibers of  axotomy .Axotomy directly damages glia, axon cytoskeleton and mitochondria, located close to the axon transection site .2+ regulation by the endoplasmic reticulum [Bcl-2 supports axon regeneration via CREB and ERK pathways, promoting Caeticulum .Glia also can directly participate in the sealing of lesion site. In some invertebrates, glia can enter the lumen of a transected axon up to its complete filling . There eThe interaction of perineuronal glia with Schwann cells plays an important role in the development and health of the PNS. Perineuronal glia form “bridges” after AT of motor nerves for the following regeneration and aim Schwann cell migration for the formation of the possibility of axon regeneration towards its target .CNS injuries often result in the formation of glial scar, which prevents axon regeneration ,64. GliaThere is also a hypothetic possibility that entire mitochondria can be transferred between neurons and glia for mutual support and mitochondrial recirculation .The mobile microglia, unlike the rest glia (macroglia) in vertebrates, develops not from the ectoderm, but from the mesoderm, and migrates to the CNS in the course of embryogenesis, henceforth implementing immune functions there . Therefo2+ is a central link of many intra- and extracellular processes in neurons and glial cells, including intercellular communication, response to trauma and post-traumatic recovery, or cell death. The disruption of Ca2+ homeostasis is one of the cell death factors [−4–10−3 M can trigger the cell death scenario, including necrosis and apoptosis [Ca factors ,11. The poptosis .2+ concentration activates a number of molecular mechanisms: calpain hyperactivation and cleavage of Na+ channels and other axon proteins, histone deacetylation, retrograde signalization via JUN-kinase, and importin pathways [2+ levels causes the release of Ca2+ and other high-and low-molecular components from the mitochondria through MPTP, which leads to the necrosis and apoptosis of nerve cells [2+ storages can act in concert with death signals, which initially do not require Ca2+, promoting the utilization of cellular components and death via apoptosis or necrosis. In addition, Ca2+-activated enzymes can cleave proteins, phospholipids, and DNA, inducing cell death and tissue damage.The increase in Capathways . The eleve cells . AT can ve cells ,71. Chan2+ regulates all stages of apoptosis. Excessive levels of cytosolic Ca2+ levels promote apoptosis via different pathways, for example, it causes Ca2+ entry to mitochondria and promotes the opening of MPTP, triggering the mitochondria-dependent apoptotic pathway. Calpains are Ca2+-dependent cysteine proteases which mediate the cleavage of some BCL-2 family apoptosis-regulating proteins BID, BCL-2, and BCL-XL. They are also responsible for the permeabilization of the outer mitochondrial membrane and the release of cytochrome c and AIF. Yet another mechanism is the activation of Ca2+-regulated phosphatase calcineurin. It dephosphorylates the BCL-2-associated cell death agonist (BAD), a proapoptotic member of BCL-2 family, thus increasing heterodimerization of BAD and BCL-2-XL and promoting apoptosis.Ca2+ plays a fundamental role in synapse activity control and memory formation, a process resulting in the activation of specific Ca2+-dependent pathways of signal transduction, and involves key protein effectors, such as calmodulin kinases, MAPK/ERK kinases, and CREB [In the brain, Caand CREB .2+ levels promotes axon regeneration after laser transection. The in vivo application of MTPT inhibitors bongkrekic acid and cyclosporin A prevented both cytochrome c release and the following activation of kaspase-3, and decreased motor neuron apoptosis. The application of an MCU inhibitor, ruthenium amino complex RU360, made similar effects [The increase in cytosolic Ca effects .2+ homeostasis, may induce apoptosis via the caspase-12 pathway, not associated with mitochondria. Additionally, Ca2+-dependent splitting and deactivation of caspase-12 by calpain is allegedly a base of apoptosis caused by ischemia and glucose deprivation [2+ is also associated with the activation of some DNA-degrading endonucleases [Moreover, ER stress induction, because of changes in ER Carivation . The incucleases .2+ is involved in the work of cytoskeleton-related factors; for example, caltubin, a mollusk protein, interacting with tubulin, promotes axon growth and decreases axon degeneration in rodents [2+-dependent proteins are involved into responses of neurons and glia to axonal injury: calpains, calmodulin, calmodulin kinases, protein kinase C, among others.Ca rodents . A numbeAT promoted calmodulin expression in the crayfish ventral nerve cord . The exp2+ through the hyperpolarization-activated plasma membrane channel HNC2. The downregulation of this channel blocks the entrance of Ca2+ into the cell, as well as AIF-regulated pathway and apoptosis [The family of protein kinase C (PKC) is a group of important signal molecules, involved in the prevention of neurodegeneration after injuries of nervous system. AT increases the level of PKC II in axotomized neurons . The inhpoptosis .+–Ca2+ exchanger in neurons, causing Ca2+ overload and necrosis, which can be prevented via calpain inhibition or expression of the exchanger, not cleavable by calpain [Calpain II can translocate to the nuclei by itself or regulate apoptosis, interacting with Bcl-2 family proteins. Calpain activation can cause permeabilization of lysosomal membranes, which results in release of toxic cathepsins into cytosol. After AT, activated calpain also cleaves and inactivates Na calpain . In neur calpain .2+, cAMP, and ERK. Regeneration in vitro also requires Ca2+, which apparently acts through protein kinases, such as ERK or PKA [2+ and cAMP promote axon regeneration in C. elegans, engaging DLK-1 kinase [2+ signaling and the activation of CREB and Erk proteins, promoting a regenerative response and neurogenesis; the expression of Bcl-2 decreases the intake and storage of Ca2+ in ER and thus leads to a stronger intracellular Ca2+ response, induced by Ca2+ influx or axotomy in Bcl-2-expressing neurons, than in controls [The growth cone formation and regeneration in various types of axons is regulated by CaK or PKA . Ca2+ an1 kinase . Bcl-2 scontrols .2+-binding protein, is overexpressed in acute neural injury and its inhibition reduces the detrimental consequences of the trauma, but, in trophic concentrations, it can promote neuro- and synaptogenesis in the CNS [S100B, a Ca the CNS .2+ concentration is required for the induction of BDNF-dependent survival of adult CNS neurons [2+ sensor 1 (NCS-1) is a survival factor for damaged neurons, mediating GDNF survival signal via the PI3K-Akt pathway [Neurotrophic factors play a significant role in protection of neurons in case of axotomy. As far back as 1995 , several neurons . The neu pathway .It is shown that astrocytes increase during an hour the level of connexin-43, a gap junction protein, and, within a day, increase the level of multifunctional GFAP protein . SimilarAT induces a complex cascade of metabolic and signal processes, aimed at counteraction to further lesion and compensation of damage inflicted, or the implementation of programmed cell death, apoptosis of autophagy . In less2+ in cell death regulation has been known for a long time. Necrotic cell death has, for a long time, been associated with intracellular Ca2+ overload, leading to membrane permeabilization and the functional collapse of mitochondria. In further specification of the signal pathways of apoptosis, it was found that Ca2+/calpain is critically involved in the work of apoptosis induction factor (AIF). Later, it was shown that Ca2+ plays an important regulatory role in other types of cell death, in particular, autophagy and anoikis [The important role of Ca anoikis .2+ inflow and calcineurin activation, induced the translocation of Bad to mitochondria and apoptosis, which can be inhibited by the co-expression of a mutant inhibitory form of calcineurin and its pharmacological inhibitors [The overexpression of anti-apoptotic protein Bcl-2  and delehibitors .Injured axons are a place of reactive oxygen production, energy supply disruption, and MPTP formation . The inhSome types of CNS injuries involve changes in mitochondrial potential and metabolism both in neurons and astrocytes linked to MPTP opening .2+ and impaired the functions of ryanodine receptors and ER Ca2+ ATPase (SERCA). As for necrosis, AT-specific pro-necrotic functions of ryanodine receptors and protein kinase C were detected [In crayfish stretch receptors, AT not only decreased firing duration  of mechanoreceptor neurons , but alsdetected . The sum2+-related homeostasis disruptions, such as ER stress [2+ is involved in AT-induced autophagy remains understudied. Autolysis  can occur if calpain or other calpain-dependent enzymes destroy lysosomal membrane, releasing cathepsins (DNAses and lipases) and reactive oxygen species, which increases the acidity of intracellular medium [2+ influx, induces sarmoptosis in sensory neurons [Despite the fact that autophagy can be both protective ,104 in aR stress ,115, ther medium . There a neurons ,116.As we said above, AT promotes the activity and expression of PMCA. At present, direct PMCA inhibitors are not easily accessible, so indirect methods of inhibiting this pump are used, for example, the increase in saline pH against SERCA inhibition .2+ release from ER. Calcium signaling in astroglia is based on combined work of IP3R store-operated Ca2+ channels (SOCC) in plasma membrane, belonging to the Orai family and acting together with STIM 1 and 2 molecules, which transmit to them the ER depletion signal [2+ concentration in remote glia.IP3 receptors (IP3R) are, together with RyR, a way of Can signal . As in t2+ uniporter (MCU), through which Ca2+ enters the mitochondria, and mitochondrial Na+–Ca2+ exchanger (NCX), releasing Ca2+ from mitochondria, regulate Ca2+ movement between mitochondria and cytosol, thus being involved in Ca2+ homeostasis and the survival of the cell. For MCU inhibition, ruthenium complexes Ru360 and Ru265 are used [Mitochondrial Caare used . There aare used .2+ concentration in RGC. To study a possible mechanism of AT-induced retrograde propagating of Ca2+ wave along glial envelope, it seems reasonable to apply gap junction inhibitors, such as arachidonic acid.There is a question about the nature of cause-and-effect processes, linking axon damage and the elevation of Ca2+ uniporter and NCX, IP3R with SOCC, and differentiated Ca2+-dependent potassium channels are a prospective direction in the research of Ca2+ mechanisms, regulating AT-induced death and the survival of RGC together with neurons. The wide spectrum of Ca2+-activated proteins also needs to be comprehensively studied for potential therapeutic targets.Thus, the analyses of the involvement of PMCA, mitochondrial CaTraumatic brain injury is one of main causes of death and disability in young and middle age, and spinal brain injury is one main causes of disability, limiting mobility in people of all ages. Peripheral nerve injuries remain a growing social-economic burden, mainly affecting the young working population. The existing methods of clinical treatment, aimed to prevent the death and degeneration of nerve cells in the first hour after a trauma, are, in general, insufficient, leaving a significant part of motor or sensory functions lost.2+ homeostasis and neuroglial interaction. To date, biomedical science has not created sufficiently effective agents and methods of treatment, aimed at both of these factors and considering their connection.One of the recovery conditions after such injures is the preservation of viability of damaged neurons and glia, which, in turn, depends on the number of factors, including Ca2+ concentrations increase or decrease both apoptosis and necrosis, and others, depending on circumstances, act more selectively, preventing or promoting a certain type of death. The important fact is that AT induces susceptibility to activation or inhibition of certain signal pathways.The death and survival of glia have substantial significance in the recovery after neurotrauma, in which, on the one hand, glia plays protective role, and on the other hand, glia should not hinder the regeneration processes. There should be some type of balance between survival (proliferation) and manageable cell death . Apparently, some ways of altering Ca2+ regulation mechanisms in AT-induced death of RGX indicate some possible directions for the search of novel and the development of existing methods of pharmacological applications for protecting neurons and glia from the consequences of neurotrauma. Nevertheless, the interplay between neurons, satellite glial cells, and Ca2+-dependent mechanisms in response to AT, despite its critical importance, especially for clinics, demands further research. Many questions remain unanswered, and many problems remain unresolved. We hope that this review will help to conceptualize the problem and, with the help of our recent data, to build a roadmap and perform new productive studies for more integrative practical understanding of what is happening with neurons and their environment after axotomy and what can be done to protect them and facilitate the subsequent regeneration and functional recovery.The already obtained data about the involvement of Ca


The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+–CaM required the cytoplasmic amino acid stretch E33–Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+–CaM.Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca The transient receptor potential yeast (TRPY) channel 1 is a member of the superfamily of transient receptor potential (TRP) cation channels. Based on their amino acid sequence similarities, the TRP proteins fall into seven subfamilies, six of which are found in mammals: canonical , vanilloid (TRP vanilloid [TRPV]), melastatin (TRP melastatin [TRPM]), mucolipin (TRP mucolipin), ankyrin (TRP ankyrin), and polycystin (TRP polycystin). The TRPN  has so far only been detected in worm, fly, and zebrafish and is proposed to be a mechanosensing channel. So far, no TRP channels have been identified in archaea or bacteria, and only few examples of TRP genes have been identified in nonland plants and fungi (Saccharomyces cerevisiae. It is encoded by the gene YVC1 (yeast vacuolar conductance 1) and forms ion channels in the yeast vacuolar membrane  required for TRPY1 activation from the cytosol  retains a residual Ca2+ dependence -dependent mechanism contributing to TRPY1 regulation. CaM is a ubiquitous and highly conserved Ca2+-binding protein conveying changes of cytoplasmic Ca2+ concentrations to numerous target proteins including ion channels. In vitro, single and multiple binding sites for Ca2+–CaM have been identified in protein fragments of the mammalian TRPs (Drosophila TRP and TRP like (TRPL)  , 23, 24.tization , TRP anktization , TRPM4 stization , and TRPtization , 29, 30.er TRPV6 , 32. Up 2+-binding motifs  is lethal  cells is inhibited by overexpressed mammalian CaM but not by the mammalian non–Ca2+-binding CaM mutant CaM. Channel inhibition was abolished by deleting a stretch of 60 amino acids within the N terminus of TRPY1, apparently representing major sites of CaM's contact with TRPY1.Here, we characterize TRPY1 as a CaA) is activated by cytoplasmic Ca2+ . The Ca2+ binding was significantly reduced when D573DDD576 were replaced by alanine residues. As positive and negative controls, 25-mer peptides of the fourth Ca2+-binding loop of mammalian CaM and its D7,9,11A, E17,18A mutant version were treated in the same way . Next, we fused the TRPY1 N and C terminus (224 and 137 amino acids) to glutathione-S-transferase (GST) and tested the fusion proteins TRPY1-N and TRPY1-C (C) for direct Ca2+ binding. GST or GST-fused yeast CaM (GST-Cmd1) and its non–Ca2+-binding mutant version Cmd1–6 , only GST-Cmd1 significantly bound 45Ca2+ . Apparently, the negative charge cluster D573DDD576 involved in Ca2+ binding of the 25-mer peptide is buried in protein domains present in the C terminus of the 137 amino acids (GST-TRPY1-C (538–675)) and thus could not be accessed by Ca2+.The yeast vacuolar TRP channel  A is actimic Ca2+ , 13, 14.ytoplasm . A 25-am  and GST-fused Cmd1 and Cmd1–6 proteins. Purified MBP-TRPY1-N and MBP-TRPY1-C were immobilized to amylose resin and incubated with GST-Cmd1 or GST-Cmd1–6 in the absence and presence of 2 mM Ca2+. Cmd1 but not Cmd1–6 significantly bound to both protein fragments of TRPY1 but only in the presence of Ca2+ (D). To confirm the latter interactions, we produced TRPY1-N and TRPY1-C with additional tags. GST-tagged TRPY1-C but not GST alone binds 6His-Cmd1 in the presence of Ca2+ . Similarly, the SUMO-TRPY1-N fusion protein strongly bound GST-Cmd1, but not GST alone, again in a Ca2+-dependent manner . The data suggest at least one interaction site of CaM at both the N and C terminus of TRPY1. This binding requires Ca2+ and a CaM molecule with intact Ca2+-binding sites.We therefore analyzed for a potential interaction of Ca of Ca2+ D. To con of Ca2+ E, left. t manner E, right.2+ reporter aequorin in wt yeast, in yeast where endogenous cmd1 was replaced by cmd1–6 and in TRPY1 knockout yeast (Δyvc1). TRPY1 activity was challenged by increasing the osmolarity in the bath solution . Overexpression of cmd1 slightly reduced the hyperosmotic shock–induced Ca2+ signal in wt cells but significantly diminished the Ca2+ signal in the cmd1–6 cells (A–C). Thus, the ability of CaM to bind Ca2+ is required to inhibit TRPY1 activity.To study the effect of CaM on TRPY1 activity in intact yeast cells, we expressed the luminescent Casolution , 13, 14.–6 cells , A and C–6 cells , A–C. Thcmd1–6 yeast vacuoles by patch clamp experiments (D). For intracellular organelles, like the vacuole, membrane potentials refer to the cytosolic side and inward currents, for example, at −80 mV, represent movement of positive charges out of the vacuole into the cytosol . Vacuoles, isolated from Δyvc1 cells, did not reveal any inward current upon application of 1 mM cytosolic Ca2+  or Ba2+ (D). The outward current  represents a vacuolar Cl− conductance . The TRPY1-independent Cl− current is slightly increased by cytosolic application of Ca2+  and even more by Ba2+ .TRPY1 is located in the yeast vacuolar membrane, and next, we recorded TRPY1 currents from wt and eriments D. For in cytosol . Applica or Ba2+ D. The ouductance , which i2+ imaging protocol to monitor TRPY1 activity in the absence and presence of increasing osmolarity of the Ca2+-containing bath solution by application of sorbitol. TRPY1 complementary DNA (cDNA) expression was induced in transfected HEK-293 cells by ponasterone A. The TRPY1 protein was detectable in induced but not in noninduced transfected HEK-293 cells (A). For Ca2+ imaging, HEK-293 cells were identified by their GFP expression, that is, green fluorescence. Application of sorbitol dose dependently caused a cytoplasmic increase of Ca2+ in TRPY1-expressing cells (B) but not in control cells only expressing GFP (C). After depleting intracellular Ca2+ stores by thapsigargin, this cytosolic Ca2+ increase in the presence of sorbitol remained (D), whereas sorbitol had no effect on HEK-293 control cells (E). Thus, TRPY1, present in the plasma membrane, is responsible for the hyperosmotic shock–induced Ca2+ influx. Application of sorbitol in the absence of extracellular Ca2+ still elicits an increase of cytoplasmic Ca2+, which equals the sorbitol-induced Ca2+ increase in the presence of extracellular Ca2+ when the plasma membrane TRPY1 is blocked by La3+, a Ca2+ flux inhibitor (F). Such Ca2+ signals are not seen in HEK-293 control cells upon stimulation with 1 M sorbitol even in the presence of extracellular Ca2+ (C). Apparently, TRPY1, transiently expressed in HEK-293 cells, is also present in the membrane of the endoplasmic reticulum (ER) and, in addition to Ca2+ influx (see aforementioned), mediates a hyperosmotic shock–induced Ca2+ release.TRPY1 retains its channel properties when expressed in HEK-293 cells , and we 93 cells A. For Cang cells B but notsing GFP C. After remained D, whereaol cells E. Thus, nhibitor F. Such Clar Ca2+ C. AppareI). Independently, another marker for the ER, ER-CFP , was expressed and compared with the expression of TRPY1-GFP (J). TRPY1-GFP and BiP-mRFP are colocalized  and TRPY1-GFP and ER-CFP exhibit the same cellular fluorescence pattern . The fluorescence images show that TRPY1 is present in ER membranes and functionally contributes to hyperosmotic shock–mediated Ca2+ release from intracellular Ca2+ stores . Infusion of 1 μM Ca2+via the patch pipette revealed whole-cell currents in TRPY1-GFP–expressing cells but not in HEK-293 control cells (A and B), showing that the TRPY1-GFP fusion protein forms a functional channel in the plasma membrane, as unfused TRPY1 does.To show the localization of TRPY1, we 3′ fused GFP cDNA to the cDNA of TRPY1. HEK-293 cells were transfected with the new cDNA to yield the TRPY1-C-GFP fusion protein (TRPY1-GFP). The cDNA of BiP, a chaperone located in the lumen of the ER, fused to mRFP (BiP-mRFP), was coexpressed as a marker for the ER I. IndepeRPY1-GFP J. TRPY1-ol cells , A and B2+ measurements (G and H). Hypertonic shock mediated a fluorescence increase, that is, volume reduction, of HEK-293 control cells in a dose-dependent manner (G), which was more transient and significantly reduced after an initial peak in TRPY1-expressing cells (H). The initial volume reduction activates TRPY1, and cation influx into the cell occurs, counteracting the hyperosmotic shock–induced volume reduction only in TRPY1-expressing cells.The cells shrink in the hyperosmotic bath solution, and we analyzed volume changes by exciting Fura-2 fluorescence at the isosbestic wavelength 360 nm and monitored the emitted light using the same filter system as for Caurements , G and Ht manner G, which ng cells H. The in2+ stores mediate the hyperosmotic shock–induced increase of cytoplasmic Ca2+ and that TRPY1 at both places may contribute to a TRPY1 function in osmoregulation.These results show that in HEK-293 cells, TRPY1 present in the plasma membrane and in the membranes of intracellular Ca2+-binding CaM mutant, carrying a D-to-A mutation in the first position of all four EF hands  mutant (A and C). Neither CaM nor CaM expression revealed any effect on the cytosolic Ca2+ upon application of 1 M sorbitol in HEK-293 control cells (B). Inhibition of TRPY1 expressed in HEK-293 cells by endogenous CaM depends, like in yeast, on Ca2+ binding, and is prevented by ophiobolin A, a CaM inhibitor (D and E). In addition, TRPY1 whole-cell currents in HEK-293 cells, activated by 1 μM Ca2+ in the patch pipette, were significantly reduced after coexpression of CaM but not the CaM mutant (F–H). We also recorded TRPY1 currents induced by hyperosmotic shock (500 mM sorbitol) after overexpression of the CaM cDNA (H–K) or activated by 1 μM intracellular Ca2+ and hyperosmotic shock (500 mM sorbitol) after cytosolic infusion of 10 μM recombinant mammalian CaM via the patch pipette (A–G). In all protocols, cytosolic CaM decreased the TRPY1 current. In contrast, coexpression of the non–Ca2+-binding CaM mutant ) had little effect on TRPY1 currents. The hyperosmotic shock–induced currents were not seen in nontransfected HEK-293 cells (L and M) and were virtually abolished in TRPY1-expressing cells in the absence of intracellular Ca2+ .Next, HEK-293 cells were cotransfected with the TRPY1 cDNA and the cDNAs of wt mammalian CaM or the mammalian non–CaEF hands . The hyp) mutant , A and Col cells B. Inhibinhibitor , D and E) mutant , F–H. WeCaM cDNA , H–K or  pipette , A–G. In93 cells , L and MThe data also show that mammalian CaM can functionally replace yeast CaM in inhibiting the activity of TRPY1. Such a functional replacement of yeast CaM by its mammalian homolog has been shown for several physiological functions in yeast , 38, 39.In vitro, Ca2+–Cmd1 is bound by both the N and C terminus of TRPY1  yielded comparable amounts of protein (A). Next, we recorded TRPY1 currents from yeast vacuoles by patch clamp experiments. Application of 1 mM Ca2+ from the cytosolic side induced inward and outward currents in vacuoles expressing wt TRPY1 (B). The current amplitudes of TRPY1-K43 48A (C) and TRPY1-K86 89 91A (D) were not distinguishable to the current amplitudes obtained from the wt channel , whereas deletion of residues 33 to 92 (TRPY-Δ33–92) including both domains increased current amplitude almost twofold (E–G). As shown in E, F, and H, replacement of wt Cmd1 by cmd1–6 had a similar effect on wt TRPY1 currents.Because most confirmed CaM-binding sites have a net positive charge, lysine residues of the major two domains at positions 43 and 48 as well as 86, 89, and 91 within the N terminus were replaced by alanine residues, and, in addition, both domains were deleted, generating TRPY1-K43 48A, TRPY1-K86 88 91A, and TRPY1-Δ33 to 92 cDNAs. The wt and the mutant cDNAs expressed in TRPY1 knockout yeast , we performed a peptide scan in order to identify Ca2+–CaM binding sites within the N and C terminus of TRPY1. We did not find clearly defined single sites but rather several CaM-binding domains (2+-activated K+ (SK) channel . However, a TRPY1 deletion mutant lacking amino acid domain R33 to Y92, covering most of the two domains identified by the peptide scan including the lysine residues, rescued the vacuolar current phenotype obtained with the non–Ca2+-binding CaM–expressing yeast strain (cmd1–6).Neither the tryptophan nor the sequence motifs of the hydrophobic patches are conserved in TRPY1, which shares less than 16% sequence identity with TRPV6 or TRPV5. We therefore replaced the lysine residues 43 and 48 (TRPY1-K43 48A) and the lysine residues 86, 89, and 91 (TRPY1-K86 89 91A) by alanine residues within those domains of TRPY1, which were most prominent in the peptide scan . We did not observe any Ca2+ binding to the complete recombinant TRPY1 C terminus K538 to E675, where the accessibility of the cluster for Ca2+ might be prevented by structural restraints (C). Maintained TRPY1 channel activity was shown after replacing D573DDD576 by asparagine residues  in the presence (2 mM Ca2+) or the absence of Ca2+ (5 mM EGTA) for 60 min at 4 °C. After washing, bound proteins were eluted with SDS sample buffer, analyzed by SDS-PAGE blotted, and detected by anti-GST antibodies.Purified MBP-fused proteins (12 μg) were immobilized to amylose resin and incubated with 12 μg of GST fusion proteins containing wt yeast CaM (Cmd1) or the non–Ca2+ (2 mM CaCl2 or 5 mM EGTA) for 60 min at 4 °C, the resins were washed, and bound proteins were eluted with SDS and analyzed by SDS-PAGE and Coomassie blue staining.GST or GST-Cmd1 (12 μg) was immobilized to glutathione sepharose, and 6His-Sumo-TRPY1-N fusion proteins were added. The Sumo moiety was inserted to increase solubility. Alternatively, immobilized GST or GST-TRPY1-C was supplemented with 12 μg of 6His-Cmd1. After incubation with or without Ca45Ca2+ binding assay of blotted proteins was performed as described , changed every 30 min, followed by incubation in the same buffer containing 1 mCi/l 45Ca2+ for 10 min. Thereafter, the membrane was rinsed with deionized water, dried at 21 °C, and exposed to a phosphorimager screen  for 12 to 24 h. The screen was scanned by a Typhoon FLA 9500 . The 45Ca binding to spotted peptides was essentially performed as described .About 20-mer peptides of the TRPY1 N and C terminus were synthesized on an Intavis ResPepSL peptide spot synthesizer and spotted onto hardened cellulose membranes at 16 nmoles per spot. The amino acid sequence was shifted by five amino acids from one spot to the next. The membranes were equilibrated with binding buffer  at room temperature for 60 min followed by incubation in the same buffer including 2 mM CaClΔyvc1) GSY1180 (MATα YVC1::TRP1) and the CaM mutant strain cmd1–6 (MATα cmd1–6) (MATα ura3 leu2 his3 trp1 ade2 can1). The cmd1–6 strain is a kind gift from Dr Trisha Davis, Seattle, WA. For Ca2+ imaging or patch clamp experiments, yeast cells were transformed with plasmids as listed in the resources table (The TRPY1 knockout yeast strain ( cmd1–6)  are isoges table . Cells wcmd1–6, and yvc1-deficient (Δyvc1) yeast cells were transformed with plasmid pEVP11-AEQ89  for 15 min at 30 °C. To remove cell walls, protoplasting buffer  was added with final concentrations of 1 mg/ml zymolyase 20T  and 150 mg/ml (324 mM) bovine serum albumin . After 45 min of incubation, spheroplasts were pelleted and resuspended in stabilizing buffer . After 2 to 3 days, the spheroplasts revealed giant vacuoles.The preparation of large vacuoles was performed as described , 13. Bries table ) were ha2, 10 mM glucose, 10 mM MES, and pH 6.8) for 2 to 5 min. Then releasing buffer was replaced by bath (cytosolic side) solution . A Zeiss Axiovert 135 microscope equipped with a 40× LD Achroplan objective (Zeiss) was used to visualize the vacuoles. Patch pipettes were pulled from glass capillaries GB150T-8P (Science Products) at a PC-10 micropipette puller (Narishige) and filled with the same solution as in the bath (see aforementioned), leading to resistances between 2 and 4 MΩ. After reaching the giga-seal, short voltage pulses  were applied to break-in and reach the whole-vacuole configuration. About 1 mM CaCl2 or BaCl2 was applied directly onto the measured vacuole via an application pipette (cytosolic side), or 1 mM CaCl2 was added into the patch pipette (vacuolar side). Currents were recorded from voltage ramps of 200 ms spanning from 150 to −150 mV from a holding potential of 0 mV applied every 2 s using an EPC-9 patch clamp amplifier (HEKA). Currents were normalized to the size (capacitance) of the vacuole and plotted as pA/pF (current densities). Inward and outward currents were extracted at −80 and 80 mV, respectively, and plotted versus time. Representative current–voltage relationships were extracted at indicated time points. Note that the membrane potentials refer to the cytosolic side, that is, inward currents at −80 mV represent movement of positive charges from the vacuole toward the cytosol  or pIND-IRES-GFP (HEK control) plus pVgRXR (Invitrogen) plasmids. About 24 h after transfection, 10 μM ponasterone A (Invitrogen) was applied to induce YVC1 expression. Fugene HD (Promega) was used as transfection reagent. Cells were used 24 to 72 h after transfection.For cell transfection, we used either the ponasterone A–inducible expression system (Invitrogen) or the bicistronic expression vector IRES-GFP , 59. HEK2+ imaging, cells were loaded in media with 5 μM Fura-2-acetoxymethylester (TEFLabs) for 30 min at 37 °C. Subsequently, cells were washed and placed into a bath chamber containing 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM Hepes, 10 mM glucose, and pH 7.2 in the presence or the absence of 1 mM CaCl2. Compounds were applied directly into the bath. The Ca2+-dependent Fura-2 fluorescence of single cells was monitored at a rate of 0.5 Hz with a dual excitation fluorometric imaging system  controlled by TILLvisION software (TILL Photonics), using a Zeiss Axiovert 200 M microscope equipped with a 20× Zeiss EC Plan Neofluar objective. Fura-2-loaded cells were excited at 340 and 380 nm for 30 ms every 2 s each, and the fluorescence emission above 450 nm was detected by an Andor iXon CCD camera. After background subtraction, positively transfected cells, identified by their GFP expression (green fluorescence), were marked as regions of interest, and their mean fluorescence at 340 nm (F340) and 380 nm (F380) excitation were computed into relative ratio units (F340/F380). To analyze hypertonic shock–mediated volume changes, cells were excited by the Fura-2 Ca2+-independent wavelength 360 nm for 30 ms every 2 s and the fluorescence emission above 450 nm was detected and normalized to the fluorescence intensity just before the hypertonic shock (F/F120s).For CapcDNA3-TRPY1-GFP) to yield TRPY1-C-GFP fusion proteins (TRPY1-GFP), the ER marker ER-CFP  and/or the ER chaperon BiP, fused to mRFP -IRES-GFP. About 24 h after transfection, cells were trypsinized and scattered on small glass coverslips (1 cm in diameter). Patch clamp experiments were performed 48 to 72 h thereafter. A Zeiss Axiovert 135 microscope equipped with a 40× LD Achroplan objective (Zeiss), a green LED (Rapp OptoElectronic) and a GFP filter set  were used to visualize and identify green cells. The bath solution contained 140 mM NaCl, 2.8 mM KCl, 3 mM MgCl2, 10 mM Hepes, and 10 mM glucose . Patch pipettes were pulled from glass capillaries GB150T-8P (Science Products) at a PC-10 micropipette puller (Narishige) and filled with pipette solution comprising 120 mM cesium glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM Hepes, 10 mM Cs-BAPTA , leading to resistances between 3 and 4 MΩ. For activation of TRPY1, the pipette solution either contained 1 μM free Ca2+ (10 mM Cs-BAPTA + 8.2 mM CaCl2) or bath solution, supplemented with 500 mM sorbitol (hypertonic shock), was applied via an application pipette directly onto the measured cell with 300 nM free Ca2+ (10 mM Cs-BAPTA + 5.7 mM CaCl2) in the patch pipette. Intracellular-free Ca2+ concentrations were calculated using webmaxc standard (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). For some experiments, 10 μM recombinant bovine  CaM (Sigma–Aldrich) was added to the patch pipette, or Ca2+ was omitted (0Cai). For tip coating, patch pipettes were dipped into sigmacote (Sigma–Aldrich). Whole-cell currents were recorded from voltage ramps of 400 ms spanning from −100 to 100 mV from a holding potential of 0 mV applied every 2 s using an EPC-9 patch clamp amplifier (HEKA). Currents were normalized to the size (capacitance) of the cell and plotted as pA/pF (current densities). Inward and outward currents were extracted at −80 and 80 mV, respectively, and plotted versus time. Representative current–voltage relationships were extracted at indicated time points.HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin/streptomycin, plated on glass coverslips (2.5 cm in diameter) and, after reaching about 80% confluency, transfected with 2+ imaging data were saved as Excel files. HEK-293 cells Ca2+ imaging data were initially analyzed in TILLvisION. Fluorescence images were captured by the AxioVision software (ZEISS) and processed in ImageJ. Patch clamp data were analyzed in Patchmaster or Fitmaster (HEKA). All data were finally transferred to IgorPro (WaveMetrics) for further analysis and graphical presentation. GraphPad Prism  was used to prepare bar graphs, scatter plots, and box plots as well as to test for statistical differences. All data were first tested for parametric or nonparametric distribution using Anderson–Darling, D'Agostino and Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov tests. Means of parametric data were compared by Student's t test for two groups and one-way ANOVA with Bonferroni's multiple comparison post hoc test for more groups. Means from nonparametric data were compared by Mann–Whitney test for two groups and Kruskal–Wallis test with Dunn's multiple comparison post hoc test for more groups. The respective tests are indicated in the legends to the figures. Parametric data are presented as bar graphs, and nonparametric data as Tukey's box and whiskers with the boxes extend from the 25th to the 75th percentile (interquartile range), and the line inside the box shows the median. Whiskers are extended to the most extreme data point that is no more than 1.5× interquartile range from the edge of the box, and outliers beyond the whiskers are depicted as dots. Error bars represent the SEM or SD as indicated in the legends to the figures. Final figures were prepared in CorelDRAW (Corel Corporation).The hydrophilicity plot was performed using Protean (DNASTAR). Luminometric CaAll relevant data are contained within the article or the supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


RYR2) gene. Mutations in RYR2 promote calcium (Ca2+) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca2+ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca2+ handling and arrhythmic events in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg‐176‐Glu (R176Q) and a mutation‐negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC‐CMs were derived using the StemdiffTM kit. Confocal Ca2+ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC‐CMs harbouring the R176Q variant demonstrated spontaneous SR Ca2+ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC50 = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca2+ handling in control iPSC‐CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca2+ handling and suppresses arrhythmogenic activity in iPSC‐CMs derived from a CPVT patient. Hence, this study confirms that this RyR2‐inhibitor represents a promising therapeutic candidate for treatment of CPVT.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal‐dominant variants in the cardiac ryanodine receptor type‐2 ( Variants in RYR2 promote diastolic SR Ca2+ leak which causes triggered activity and can initiate arrhythmias seen in CPVT.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life‐threatening inherited cardiac arrhythmia syndrome that can lead to sudden cardiac arrest and death at a very high rate if left untreated. It is characterized by polymorphic and/or bi‐directional ventricular tachycardia (VT) that is triggered by β‐adrenergic stimulation associated with vigorous exercise or emotional stress.+ channel blocker that also normalizes RyR2 activity—has shown potential in CPVT patients but also has a black box warning for patients with any kind of structural heart disease.Although CVPT is one of the most lethal forms of inherited cardiac disease, current treatment options remain limited. β‐adrenoreceptor blockers only control symptoms and suppress arrhythmias in about 30%‐50% of patients.Several classes of drugs have been developed that target mutant or dysfunctional RyR2 channels.2+ leak associated with CPVT in the R176Q mouse model.+ or IKr channels within the effective concentration range used to inhibit RyR2 channels and has a favourable plasma stability profile (data not shown). In addition, tetracaine derivatives appear to work for all gain‐of‐function RyR2 variants, which represent the vast majority (>95%) of all known CPVT‐causing variants (>150 described to date). It remains unclear, however, whether a compound like EL20 also normalizes CPVT‐mutant RyR2 in human cardiac myocytes. Therefore, we developed induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) from a CPVT patient carrying the same R176Q variant in RYR2. Confocal imaging revealed that EL20 effectively suppresses the enhanced Ca2+ spark frequency observed in iPSC‐CMs from this CPVT patient. These findings suggest that this RyR2‐inhibitor represents a promising therapeutic candidate for treatment of CPVT patients.We recently demonstrated that tetracaine derivative EL20 can normalize aberrant SR Ca22.1All studies were approved by the Baylor College of Medicine IRB and follow the Declaration of Helsinki guidelines. Subjects were evaluated by a paediatric electrophysiologist and a history, physical examination, echocardiogram, electrocardiogram and ancillary testing were performed. Following written receipt of informed consent, whole blood was obtained from the proband and kindred.2.2TM CM differentiation kit (Stemcell Technologies) according to the company protocol (Document #DX21496). See iPSCs were differentiated into iPSC‐CMs using the STEMdiff2.3TM Cardiomyocyte Dissociation Kit . See Spontaneously beating CMs were dissociated using the STEMDiff2.4The tetracaine derivative, 2‐(diethylamino)ethyl 4‐butlamino)‐2methoxybenzoate, also known as EL20, was synthesized using a two‐step synthesis route as previously described by Klipp et al.2.52+ sparks were recorded in line‐scan mode using a LSM880 confocal microscope (Carl Zeiss). CaSpF was assessed with ImageJ software using SparkMaster plugin. See For drug assay studies, iPSC‐CMs were incubated with drug (concentration range 0.5 nmol/L‐5 μmol/L) for 30 minutes at 37°C. Ca2.6t test or ANOVA, after performing the D’Agostino‐Pearson normality test for normal data distribution. For multiple group comparison, two‐way ANOVA followed by Tukey's post‐test was used. P < .05 was considered statistically significant.Results are expressed as mean ± SEM. For clustered data in which continuous variables were observed, the generalized estimating equation was used via SPSS version 24 (IBM) or Prism 8 (GraphPad) using an unpaired Student's 33.1RYR2 . The CPVT proband is currently being treated with a β‐blocker and has an ICD. Her father (41‐year‐old) is an unaffected family member and is considered a healthy, mutation‐negative control.The patient was previously a seemingly healthy female with a family history of sudden cardiac death Figure . Her mat3.22+ handling markers  were extracted and subsequently reprogrammed into iPSCs using Sendai viruses Figure A‐D. Bothrs Table C. Immunors Table D.3.32+ imaging to characterized intracellular Ca2+ handling in dissociated iPSC‐CMs that exhibited a ventricular‐type morphology. About 50% of iPSC‐CMs exhibited non‐evoked, spontaneous rhythmic beating without electrical stimulation  consistent with being arrhythmic . The amplitudes of non‐evoked Ca2+ transients were similar between the groups   . Finally, the amplitude of the Ca2+ transients    phenotypes.n Figure . The avec Figure . In addi) Figure . The SR ) Figure . Next, w) Figure ; the rem) Figure  and tota) Figure  was simi3.4P = .002). 53% of the iPSC‐CMs with the R176Q variant exhibited abnormal SR Ca2+ release events during the final phase of SR Ca2+ reuptake, compared to 0% of the control cells (P < .05)    was added to the iPSC‐CMs. Addition of ISO led to a positive chronotropic response compared to baseline conditions, as typically seen in healthy iPSC‐CMs.) Figure . The amp) Figure . The SR ) Figure .2+ transients following the 1‐Hz pacing train  was similar amongst the two groups  compared with control iPSC‐CMs , suggesting that the oscillating SR Ca2+ release events interfere with SR Ca2+ refilling.About 75% of the iPSC‐CMs that did not spontaneously beat did generate SR Can Figure . While ss Figure . However3.52+ sparks were recorded using line‐scan mode on a LSM880 confocal microscope to determine the diastolic activity of RyR2 in iPSC‐CMs following a 1‐Hz conditioning pacing train. Because spontaneous contractions in the non‐evoked iPSC‐CM population can potentially confound diastolic Ca2+ releases, Ca2+ imaging was done only under evoked pacing conditions to profile the drug responses of EL20 in vitro. In the presence of 100 nmol/L ISO, iPSC with the R176Q mutation exhibited a higher Ca2+ spark frequency (CaSpF) (3.6 ± 0.45 sparks/100 μm/s) compared with iPSC from the control  . In contrast, in R176Q iPSC‐CMs, EL20 significantly reduced the CaSpF by 78% .Ca) Figure . Next, a3.6−9 up to 10−4 mol/L  of EL20 is 8.2 × 10−8 mol/L. Hence, EL20 was found to inhibit excessive SR Ca2+ release at low nanomolar concentrations that are clinically relevant.To get a better sense of what drug concentrations are needed to inhibit mutant RyR2 channels, we assessed the effects of different doses of EL20 on CaSpF in evoked iPSC‐CMs derived from the CPVT proband. The dose‐response curve, which is based on inhibition of the CaSpF, was determined at concentrations ranging from 10L Figure . Results3.72+ release through mutant RyR2, it is possible that this drug has unwanted effects on systolic SR Ca2+ transients consequently leading to unwanted side effects.2+ transient amplitude induced by field stimulation at 1‐Hz in iPSC‐CMs  compared to control + vehicle . Importantly, EL20 also did not alter the Ca2+ transient amplitude in R176Q iPSC‐CMs (F/F0: 1.7 ± 0.20) compared to R176Q + vehicle . Interestingly, treatment with EL20 prevented to occurrence of pacing‐evoked Ca2+ oscillations  and could be more effective compared to beta blockers that work upstream in the beta‐adrenergic receptor signalling pathway that leads to RyR2 phosphorylation and exacerbation of SR Ca2+ leak during stress or exercise.2+ leak at multiple levels in the signalling pathway. In conclusion, our patient‐derived iPSC‐CM model offers a promising platform for further research into the pathophysiological mechanisms of CPVT, as well as a safe tool for screening and optimizing drug therapy using novel RyR2 inhibitors.We found that iPSC‐CMs derived from a CPVT patient carrying RyR2 variant R176Q display irregular spontaneous CaRMS and XHTW are founding partners of Elex Biotech, a start‐up company that developed drug molecules to target ryanodine receptors for treatment of cardiac arrhythmias. Other authors have no conflicts related to this study.Tarah Word: Data curation (lead); Formal analysis (lead); Methodology ; Writing–original draft (lead). Ann Quick: Investigation (supporting); Writing–review and editing (supporting). Christina Miyake: Formal analysis (supporting); Investigation (supporting). Mayra Shak: Methodology (supporting). Xialo Pan: Methodology (supporting); Resources (supporting). Jean Kim: Methodology (supporting); Supervision (supporting). Hugh Allen: Supervision (supporting). Martha Sibrian‐Vazquez: Resources (supporting). Robert Strongin: Supervision (supporting); Validation (supporting); Writing–review and editing (supporting). Andrew Landstrom: Resources (supporting); Supervision (supporting); Writing–review and editing (supporting). Xander H. Wehrens: Conceptualization (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.