ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca Calcium deposition in the vascular wall involves osteo-/chrondrogenic reprogramming of vascular smooth muscle cells (VSMCs)8. The signaling includes up-regulation of the transcription factor NFAT5 (nuclear factor of activated T cells 5)12. NFAT5 was originally cloned as tonicity responsive enhancer binding protein (TonEBP) stimulated by hyperosmotic cell shrinkage14 and subsequently been shown to be enhanced in several disorders, such as diabetes15, inflammation16 and CKD12.Compromised renal elimination of phosphate leads in chronic kidney disease (CKD) to increase of phosphate concentration in plasma and tissues which in turn triggers vascular calcification leading to cardiovascular events and the respective increases of morbidity and mortality17. SGK1 up-regulates the transcription factor NFκB (nuclear factor κB)18. NFκB triggers transcription of the Ca2+-channel ORAI1. Following depletion of intracellular Ca2+-stores ORAI1 is activated by Ca2+ sensing STIM 18. Opening of ORAI1 by STIM leads to the so-called store operated Ca2+-entry (SOCE)18, SOCE is a powerful mechanism activating blood platelets20. Genetic or pharmacological knockout of SGK1 down-regulates ORAI121, blunts platelet activation23 and thus counteracts thrombosis23 and arteriosclerosis24. SGK1 and thus SGK1-dependent up-regulation of ORAI1 expression is stimulated by ischemia, oxidative stress, hyperglycemia, advanced glycation end products (AGEs) and several mediators including glucocorticoids, mineralocorticoids, transforming growth factor beta (TGFβ), interleukin 6 (IL-6), platelet-derived growth factor (PDGF), thrombin and endothelin25. By stimulating ORAI1 expression, upregulated SGK1 may increase the risk of thrombo-occlusive events in diabetes mellitus, inflammation, and chronic kidney disease25.NFAT5-regulated genes include SGK1 (serum and glucocorticoid inducible kinase 1)27. Megakaryocytes reorganize their cytoplasm into long proplatelet extensions that release platelets into the circulation27. Consequently, the proteins expressed in megakaryocytes are expected to be transferred into circulating blood platelets27. Recent observations revealed a powerful SGK1 dependent stimulation of ORAI1 expression by NFAT5 overexpression in megakaryocytes28.Platelets are released into the blood stream by megakaryocytes, which differentiate from hematopoietic progenitor cells in the bone marrowIn view of those observations we hypothesized that the phosphate-donor ß-glycerophosphate mimicking enhanced extracellular phosphate concentration may up-regulate the expression of NFAT5 in megakaryocytes, and that NFAT5 enhances the expression of SGK1, ORAI1 and STIM1 and/or STIM2. Considering that the abundance of the respective proteins in circulating platelets is a function of protein synthesis in megakaryocytes, enhanced extracellular phosphate may up-regulate the expression of NFAT5, SGK1, ORAI1 and STIM1 and/or STIM2 in circulating blood platelets. To the best of our knowledge, however, nothing is hitherto known on the contribution of NFAT5 to the regulation of megakaryocyte or platelet function in CKD patients.The present study thus explored whether NFAT5, SGK1, ORAI1, ORAI2, ORAI3 STIM1 and/or STIM2 expression in megakaryocytes is sensitive to phosphate-donor ß-glycerophosphate and altered in patients with CKD incl. dialysis-dependency.17 which in turn activates transcription factor NFkB with upregulation of ORAI and STIM isoform expression18, the present study explored whether the phosphate donor ß-glycerophosphate modifies the transcript levels of the transcription factor nuclear factor of activated T cells 5 (NFAT5), of the NFAT5-regulated serum & glucocorticoid inducible kinase 1 (SGK1), of the SGK1-sensitive Ca2+ release activated ion channels ORAI1, ORAI2 and ORAI3 as well as of the ORAI activating Ca2+ sensor isoforms STIM1 and STIM2. As illustrated in Fig. 2+ entry into UMR106 bone cells29.As the transcription factor NFAT5 is known to upregulate expression of SGK12+ signaling, Fura2 fluorescence was utilized to determine the cytosolic Ca2+ activity ([Ca2+]i). The 340 nm/380 nm ratio reflecting [Ca2+]i was, prior to store depletion, similar in ß-glycerophosphate treated  and in untreated  megakaryocytes. For determination of SOCE, cells were exposed to thapsigargin (1 µM), a sarco-/endoplasmic reticulum Ca2+/ATPase (SERCA) inhibitor, and Ca2+-free solutions to deplete intracellular Ca2+ stores. In the following extracellular Ca2+ was re-added in the continued presence of thapsigargin to quantify SOCE from the increase of [Ca2+]I  in multiple cell types30 including platelets20 and megakaryocytes22. To the best of our knowledge, an effect of phosphate or of phosphate donor ß-glycerophosphate on ORAI, STIM and their isoforms has never been shown before in megakaryocytes or platelets.The present study discloses a novel effect of the phosphate donor ß-glycerophosphate in megakaryocytes, i.e. the upregulation of ORAI1, ORAI2, ORAI3, STIM1 and STIM2 expression. ORAI1 is a Ca17, which is known to trigger the degradation of the inhibitor protein IκBα thus allowing nuclear translocation of the transcription factor NFκB18. Genes up-regulated by NFκB include ORAI118.The effect of phosphate-donor ß-glycerophosphate is disrupted by pharmacological inhibition of SGK1 and is thus presumably due to upregulation of SGK1 by NFAT5. NFAT5 has previously been shown to increase the expression of SGK112, but not in megakaryocytes or blood platelets.In view of the upregulation of NFAT5, SGK1, ORAI1, STIM1 and STIM2 expression in megakaryocytes by the phosphate donor ß-glycerophosphate, the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 were expected to be enhanced in platelets from patients with impaired kidney function. As a matter of fact, the transcript levels of each, NFAT5, SGK1, ORAI1, ORAI2, STIM1and STIM2 were significantly higher in platelets isolated from patients with impaired kidney function than in platelets isolated from control volunteers. As shown for NFAT5 and ORAI1, the increase of transcript levels is paralleled by the respective increase of protein abundance. NFAT5 expression has been shown to be upregulated by patients with advanced CKD incl. dialysis-dependency in other cell types31. The presently observed stimulation of ORAI1 and STIM2 could thus contribute to the known high risk of cardiac infarction and stroke in CKD patients33. Excessive activation of blood platelets is well known to enhance the risk of cardiac infarction and stroke34. Additional effort is needed to define the contribution of phosphate-sensitive ORAI1 and STIM2 expression in blood platelets to the enhanced cardiovascular risk in patients with advanced CKD.The upregulation of ORAI1 and STIM2 in platelets of CKD patients is expected to sensitize the platelets for activators11, diabetes mellitus15 and inflammatory disease16. In those conditions the enhanced NFAT5 expression may lead to stimulation of SGK1 expression with subsequent upregulation of ORAI1, STIM1 and STIM2 in megakaryocytes and sensitization of blood platelets to activating stimuli. The signalling may again involve SGK1-sensitive degradation of the inhibitor protein IκBα thus allowing nuclear translocation of the transcription factor NFκB.As shown for other cell types, NFAT5 is up-regulated in further clinical disorders, such as dehydration39 and STIM44 isoforms accomplish store operated Ca2+ entry (SOCE) in a myriad of cell types. Alterations of cytosolic Ca2+ activity in turn contribute to the regulation of diverse cellular functions including excitation, exocytosis, migration, cell proliferation and cell death49. Notably, ORAI1 and STIM1 are expressed in lymphocytes thus participating in the orchestration of immune response and inflammation51. The overexpression of ORAI1 and STIM1 in tumor cells contributes to cancer growth56. Future experimental effort will be required to define the pathophysiological impact of phosphate sensitive ORAI and STIM expression on inflammation and malignancy in CKD.ORAI1 expression and thus SOCE may be sensitive to phosphate or ß-glycerophosphate in other cell types. The ORAI29. Here we demonstrate that the phosphate donor ß-glycerophosphate upregulates FGF23 in megakaryocytes. In view of the putative influence of SOCE on FGF23 transcription in UMR106 cells29, it is tempting to speculate that the observed signaling contributes to the phosphate-induced FGF23 release from bone. Whether ORAI1 expression and function is sensitive to phosphate in UMR106 cells remains, however, to be shown.Genes previously shown to be upregulated by NFAT5, SGK1, ORAI1, ORAI2, STIM1 and STIM2 in UMR106 cells include FGF232+ entry (SOCE) into megakaryocytes  with impaired kidney function and 12 volunteers with normal kidney function  have been enrolled in the study. The patients were recruited from the Department of Internal Medicine, University Hospital Tuebingen. The study was performed in accordance to the approval by ethics committee of the University of Tübingen (270/2011BO1) and in accordance with the Declaration of Helsinki. Informed written consent was provided by both, volunteers and patients. Clinical details of the patients are compiled in Table 3, 150 mmol/liter NaCl, 0.36 mmol/liter NaH2PO4, 5.5 mmol/liter glucose, and 1 mg/ml BSA, pH 6.5). The platelets were resuspended after removing supernatant, in 200 µl of Tyrodes/HEPES buffer  and used for RNA/protein isolation. ~98% purity of the platelet preparation was obtained as determined by a Haematology Analyser .Venous blood was drawn into tubes containing acid/citrate/dextrose buffer, from the antecubital vein of volunteers and patients with impaired kidney function. Platelet-rich plasma (PRP) was removed by centrifugation at 430 × g for 20 min. PRP was centrifuged at 900 × g for 10 min after adding Tyrodes/HEPES buffer  for 24 hours in the absence and presence of ORAI1 inhibitor MRS1845 (10 µM) . In the analysis of vascular calcification, phosphate donor ß-glycerophosphate is widely used as substitute for phosphate and well established stimulator of tissue calcification59.Human megakaryocytic cells (Meg01) from ATCC  were cultured in RPMI 1640  containing 10% FBS and 1% Penicillin/Streptomycin in humidified incubator at 37 °C and 5% CO64. DNAse digestion was performed to avoid DNA contamination and was followed by reverse transcription using random hexamers  and GoScript™ reverse transcription system . Real-time polymerase chain reaction (RT-PCR) amplification of the respective genes were set up in a total volume of 20 µl using 40 ng of cDNA, 500 nM forward and reverse primer and 2x GoTaq® qPCR Master Mix  following manufacturer’s protocol. Cycling conditions were as follows: initial denaturation at 95 °C for 2 minutes, followed by 40 cycles of 95 °C for 15 seconds, 55 °C for 15 seconds and 68 °C for 20 seconds. For amplification the following primers were used (5′- > 3′ orientation) ):To determine transcript levels of NFAT5, ORAI1, ORAI2, ORAI3, STIM1, STIM2 and FGF23 total RNA was extracted according to manufacturer’s instructions with TriFast NFAT5fw: GAGCAGAGCTGCAGTATrev: AGCTGAGAAAGCACATAGGAPDH:fw: TGAGTACGTCGTGGAGTCCACrev: GTGCTAAGCAGTTGGTGGTGORAI1:fw: CGTATCTAGAATGCATCCGGAGCCrev: CAGCCACTATGCCTAGGTCGACTAGCORAI2:fw: CAGCTCCGGGAAGGAACGTCrev: CTCCATCCCATCTCCTTGCGORAI3:fw:CTTCCAATCTCCCACGGTCCrev:GTTCCTGCTTGTAGCGGTCTSGK1:fw:TTCCTATCGCAGTGTTTCAGTTCTTrev:CACACTCACACGACGGTTCACSTIM1:fw: CCTCGGTACCATCCATGTTGTAGCArev: GCGAAAGCTTACGCTAAAATGGTGTCTSTIM2:fw: CAAGTTGCCCTGCGCTTTATrev: ATTCACTTTTGCACGCACCGFGF23:fw: ATGAGCGTCCTCAGAGCCTArev: AGACGTCGTACCCGTTTTCCMelting curves were analysed to confirm PCR product specificity. CFX96 Real-Time System  was used to perform real-time PCR amplifications and all experiments were done in duplicate. The house-keeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified to standardize the amount of sample RNA.64. After isolation of platelets from control donors and patients with impaired kidney function, platelets were centrifuged for 5 minutes at 600 g and 4 °C. The pellet was washed with ice cold PBS and suspended in 50 μl ice-cold RIPA lysis buffer  containing Protease Inhibitor Cocktail . After centrifugation  the supernatant was taken to determine protein concentration using the Bradford assay . For Western blotting 50 µg of protein were electro-transferred onto a nitrocellulose membrane after electrophoresis using 12% SDS- PAGE and blocked with 5% milk in TBST at room temperature for 1 h. The membranes were incubated with primary anti-NFAT5 antibody , anti-ORAI1 antibody  and anti-GAPDH antibody  at 4 °C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase  for 1 h at room temperature. Protein bands were detected after additional washes (TBST) with an ECL detection reagent . For densitometry image analysis, western blots were scanned and analyzed by ImageJ software , and the results are shown as the ratio of total protein to GAPDH normalized to the control group. Protein-Marker VI  was used as reference to assign the right protein size.Protein abundance of NFAT5, ORAI1, and GAPDH was determined by Western blotting2+ concentration ([Ca2+]i), Fura-2/AM fluorescence was utilized65. Cells were preincubated for 15–30 minutes with Fura-2/AM  at 37 °C and excited alternatively at 340 nm and 380 nm in an inverted phase-contrast microscope  through an objective (Fluor 40×/1.30 oil). At 505 nm, the emitted fluorescence intensity was recorded. Data (6/minute) were acquired using a computer software Metafluor . To estimate cytosolic Ca2+ activity, a ratiometer (340 nm/380 nm) based analysis was employed. SOCE was determined following extracellular Ca2+ removal causing store depletion and subsequent Ca2+ re-addition in constant presence of SERCA inhibitor thapsigargin . For quantification of Ca2+ entry, the slope (delta ratio/s) and peak (delta ratio) were determined following re-addition of Ca2+. Experiments were performed with Ringer’s solution containing (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 32 HEPES, 2 Na2HPO4, 1.2 MgSO4, 5 glucose, pH 7.4. Ca2+-free conditions were achieved by using Ca2+-free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4.To determine cytosolic Can represents the number of independent experiments (i.e. in fluorescence experiments the number of dishes measured). Patient data are shown in scatter plots to illustrate the scatter of the data between patients. All data were tested for significance using unpaired t-test (Student’s t-test) or ANOVA. Results with p < 0.05 were considered statistically significant.Data are provided as means ± SEM, 66.The study was approved by the ethics committee of the University of Tuebingen (270/2011BO1) and has been executed in accordance with the Declaration of Helsinki. Both, volunteers and patients provided informed written consent. The data have been presented at a conferencesupplementary information.


GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) are associated with autosomal dominant cone/cone-rod (CORD) dystrophies. The nature of the inheritance pattern implies that a pool of normal GCAP proteins is present in photoreceptors together with the mutated variant. To assess whether human GCAP1 and GCAP2 may similarly regulate the activity of the retinal membrane guanylate cyclase GC-1 (GC-E) in the presence of the recently discovered E111V-GCAP1 CORD-variant, we combined biochemical and in silico assays. Surprisingly, human GCAP2 does not activate GC1 over the physiological range of Ca2+ whereas wild-type GCAP1 significantly attenuates the dysregulation of GC1 induced by E111V-GCAP1. Simulation of the phototransduction cascade in a well-characterized murine system, where GCAP2 is able to activate the GC1, suggests that both GCAPs can act in a synergic manner to mitigate the effects of the CORD-mutation. We propose the existence of a species-dependent compensatory mechanism. In murine photoreceptors, slight increases of wild-type GCAPs levels may significantly attenuate the increase in intracellular Ca2+ and cGMP induced by E111V-GCAP1 in heterozygous conditions. In humans, however, the excess of wild-type GCAP1 may only partly attenuate the mutant-induced dysregulation of cGMP signaling due to the lack of GC1-regulation by GCAP2.Missense mutations in the  A variety of protein-protein, protein-nucleotide and protein-ion interactions finely regulates the cascade2, whose dynamics depend on the interplay between Ca2+ and cyclic guanosine monophosphate (cGMP), the second messengers involved in the signaling process4. The core of the Ca2+/cGMP signaling unit resides in the complex formed by the membrane retinal guanylate cyclase  and guanylate cyclase-activating proteins (GCAPs). This supramolecular machinery ensures a precise control of the cGMP synthesis by GC1 as a consequence of the level of intracellular free Ca2+, which drops from few hundred nanomolar in the dark to below 100 nM in the light6. Several GCAP isoforms exist in various species. In mouse7 and in bovine9 photoreceptors both GCAP1 and GCAP2 have been shown to regulate the target GC1, while in humans so far only GCAP1 has been directly shown to regulate the activity of GC111.The absorption of photons by visual pigments in retinal rod and cone cells triggers a complex signaling cascade known as phototransduction, which results in the electrical response of the cell and generates the visual stimulus2+ concentration and adopt specific conformations required for controlling the activity of the target GC1, per se unable to respond to Ca2+. At high [Ca2+], GCAPs adopt a Ca2+-loaded state that inhibits GC1 activity, but following the drop in intracellular [Ca2+] during the light activation of the cascade, they switch to a Mg2+-bound conformation15 that stimulates GC1 to rapidly restore dark-adapted cell conditions by enhancing the synthesis of cGMP16.GCAPs are neuronal calcium sensors that detect subtle changes in CaGUCA1A gene encoding GCAP1, which have been associated with cone (COD) or cone-rod (CORD) dystrophies, severe forms of retinal dystrophy characterized by central vision loss, impaired color vision and photophobia19. No COD/CORD mutation in the gene encoding GCAP2 is known to date. When studied in reconstituted in vitro systems, most of the point mutations in GUCA1A resulted in GCAP1 variants that constitutively activate the GC1 over the physiological range of [Ca2+]25. The Y99C-GCAP1 mutation was the first one to have been discovered24, moreover it was the first one to be studied in a transgenic mouse line26. Other transgenic mouse lines were then generated to study similar COD/CORD-related phenotypes associated with mutations in GCAP1, namely the E155G-GCAP127 and L151F-GCAP128 models. The in vivo studies showed that the COD-related mutant caused photoreceptor degeneration due to an elevated [Ca2+] in the rod outer segment. However, rod photoresponses from the Y99C-GCAP1 mice showed relatively little changes especially at bright flashes, indicating a partly preserved Ca2+-regulated cGMP synthesis. Interestingly, the rate of photoreceptor cell loss increased with the level of Y99C-GCAP1 expression26.To date, 20 missense mutations have been found in the 2+], however the two GCAPs differ from one another in some features: (i) GCAP1 has a slightly lower affinity for Ca2+ compared to GCAP212; (ii) GCAP1 triggers the activation of GC1 earlier than GCAP2, that is at dimmer light conditions and/or when intracellular Ca2+ is still relatively high3; (iii) both GCAPs are myristoylated, but the myristoylation seems to exert a more prominent role for GCAP1 compared to GCAP2 in GC1 activation9. Moreover, in bovine cells the cellular concentration of GCAP1 and GCAP2 sums to the cellular concentration of GC19. While it is clear that both GCAPs can regulate GC1 over the narrow physiological window of intracellular Ca2+, quite different mechanisms have been proposed for the regulation. Some studies support distinct binding interfaces between GC1 and GCAP1/GCAP2, the first GCAP being located at the juxtamembrane region and the second prevalently bound to the catalytic-C terminal domain31, thus allowing the binding of GCAP1 and GCAP2 to either the same or distinct GC1 molecules, but always in different interfaces On the other hand, other studies found a partly overlapped GC interface for GCAP1 and GCAP233, suggesting a competition between GCAP1 and GCAP2 for GC1 activation in mouse cones34 as well as in rods. Overall, GCAPs seem to operate in a “calcium-relay” mode, thus acting simultaneously on either the same or distinct GC1 molecules to induce gradual responses in terms of cGMP synthesis triggered by small changes in intracellular [Ca2+]35. Their slightly different GC1-regulatory properties and Ca2+-sensitivity would therefore be needed to achieve the complex temporal dynamics necessary for finely tuning the enzymatic activity of GC1 over the broad range of light stimuli typical of photoreceptors.For bovine and murine photoreceptors, it is well established that GCAP1 and GCAP2 are both capable of activating and inhibiting the target GC1 at similar levels in the physiological range of . The discovery of the highly cooperative negative feedback mechanism mediated by Ca2+ on the GC1 dates back to the late 80’s38, and yet the fine mechanisms of its regulation are not completely understood. It has been established that the GCAP1/2-mediated Ca2+ feedback on GC1 activity is the only one that occurs at very dim light intensities, corresponding to the single photon response39. By integrating quantitative information arising from assays performed with recombinant systems and numerical simulations, we sought to clarify some mechanisms that are apparently perturbed in cases such as the severe CORD form recently associated with the E111V mutation in GCAP110. To investigate the role of each GCAP variant, namely WT-GCAP1, E111V-GCAP1 and WT-GCAP2 in GC1 (dys)regulation, we performed biochemical assays both in the presence of individual GCAPs and in the co-presence of the variants, and measured quantitative parameters to describe the Ca2+-dependence of the cGMP synthesis by GC1 and the apparent cooperativity of the process.Regulation of the GC1 activity by Ca2+ by switching to a different conformation that showed the typical increase in electrophoretic mobility  and GCAP2 were heterologously expressed and purified at high purity levels. All GCAP variants responded to Caity Fig. , as obseieu Fig. , thus al2+, as observed in previous studies11, GCAP2 was unable to exert any regulation of GC1 as in both low and high Ca2+ conditions the levels of cGMP synthesis were unaltered with respect to those of the control  with respect to the sole presence of GCAP1, thus indicating that the presence of GCAP2 only slightly interferes with the activity of GCAP1, which was still capable of regulating GC1. No GCAP2-induced activation of GC1 was observed even when the experiments were performed at higher (12 mM) Mg2+ , WT-GCAP1 correctly switched from GC1-inhibitor to GC1-activator  and the cooperativity of the process was high  and a significantly lower cooperativity  . Therefore, although the switch was still non-optimal, the presence of 3-fold excess WT-GCAPs seems to slightly attenuate the dominant and detrimental effect caused by the E111V-GCAP1 mutation.Since human GCAP2 does not activate GC1, we sought to investigate whether the observed partial restoring capability of WT-GCAP1 to restore GC1 activity in the presence of E111V-GCAP1 could even increase in the presence of extra WT-GCAP1. The assays were performed with 3-fold excess of WT-GCAP1, while keeping the concentration of E111V unaltered with respect to the previous conditions. Interestingly, a statistically significant decrease of IC50) for GC110, therefore it seems unlikely that the cause of the still dominant effect of the mutant in a situation of 3-fold excess of WT be attributed to the preferential formation of a complex with the GC1. A more realistic explanation could be the existence of different equilibria between the oligomeric states of WT-GCAP1 and E111V-GCAP1, which could result in different amounts of monomeric GCAP available for GC regulation. GCAP1 is known to form dimers45 and under conditions mimicking the physiological ones E111V-GCAP1 was also found to be dimeric10. We cannot exclude that in the conditions of our in vitro assays the tendency of WT-GCAP1 to form dimers enhanced at the higher concentrations corresponding to the 3-fold excess experiments, de facto limiting the availability of monomers that could further improve the restoration of the GC1 regulation towards a physiological behavior.The observed results of GC1 activity in the presence of different amounts of GCAP1 and its E111V variant are not straightforward to interpret. Human WT-GCAP1 and E111V-GCAP1 have very similar apparent affinity , in line with the Ca2+-relay mechanism35. In the case of WT/E111V heterozygous mouse, however, the unbalance in the contributions became apparent under dim light conditions, where GCAP1 variants dominated, while GCAP2 was still capable of shaping the rate at brighter flashes  was added. After the induction with 1 mM IPTG, cells were let grown for 4 hours at 37 °C. Both WT and E111V-GCAP1 as well as GCAP2 were purified from inclusion bodies. After protein denaturation by 6 M guanidinium hydrochloride and refolding steps by dialysis, two subsequent chromatographic steps (size exclusion and anionic exchange chromatography) were performed. Proteins purity was tested by Polyacrylamide Gel Electrophoresis in the presence of Sodium-Dodecyl-Sulfate (SDS-PAGE) and was found to be at least 95%  corresponding to the cDNA of human GCAP1 (Uniprot: P43080) and GCAP2 (Uniprot: Q9UMX6) were cloned into pET-11a(+) vectors using NdeI and NheI as restriction site. Mutagenesis, protein expression and purification of myristoylated proteins were performed as described previously51 by transfecting HEK293 flp cells with pIRES-eGFP plasmid using Turbofect reagent. Transfected clones were selected based on the resistance to geneticin (G418). Cells were cultured in DMEM media containing fetal bovine serum (10% v/v), streptomycin (100 μg/mL), penicillin (100 units/mL) and geneticin (500 μg/mL). At 90% confluence the cells were harvested, washed with sterile PBS and stored at −80 °C. To perform enzymatic assays on isolated membranes cells pellets were suspended in lysis buffer  and incubated for 20 min on ice. After 15 up-and-down cycles with a 1 mL syringe on ice, cells were centrifuged for 20 min at 10000 rpm. The obtained pellets were suspended in 50 mM HEPES pH 7.4, 50 mM KCl, 20 mM NaCl, 1 mM DTT.A stable cell line expressing human guanylate cyclase isoform 1 (GC1) was obtained as explained previously2+, obtained using 10 mM K2H2EGTA and K2CaEGTA buffers51 respectively . Basal GC1 activity was assessed as internal control by replacing GCAPs with equal amount of the buffer used for protein storing. Then, the enzymatic activity as a function of [Ca2+] was measured as in previous work10, with minor changes explained in the following. Several combinations of GCAPs were tested, namely: (i) 10 µM WT-GCAP1; (ii) 5 μM WT-GCAP1 + 5 μM E111V-GCAP1; iii) 15 μM WT-GCAP1 + 5 μM E111V-GCAP1. Data for E111V-GCAP1 alone are referred to our previous publication10. Ca2+ concentration in each assay was carefully determined by combining the same K2H2EGTA and K2CaEGTA buffers previously mentioned. The assay consisted in incubating for 5 min at RT the diluted proteins with a fixed amount of extracted membranes, then reaction buffer  was added and the samples were incubated for 5 min at 30 °C. The reaction was blocked by the addition of 50 mM EDTA and incubating at 98 °C for 5 min. After centrifugation at 13000 rpm the supernatant was loaded in a C18 reverse phase column  previously equilibrated with 5 mM KH2PO4. Following a previously established protocol10 on an HPLC apparatus (Jasco) the cGMP concentration was determined by acetonitrile gradient. Four to 6 repetitions of each assay were performed. Data have been normalized to the total protein content present in membranes used for the assay  and on the minimum and maximum GC-activity. IC50 and Hill coefficient (hc) values were determined for each assay by curve fitting to a 4-parameter Hill function (Sigma Plot 12.5) and mean, standard deviation and standard error of the mean (s.e.m.) were determined. All data distributions passed the Shapiro-Wilk normality test and the observed parameters IC50 and hc were compared with one another by one-tailed t-test (SigmaPlot 12.5), rejecting in all cases the null hypothesis (p < 0.05).First, GC1 activity was assessed incubating 10 μM GCAP1 or GCAP2 or a combination of both (5 μM GCAP1 + 5 μM GCAP2) in the absence (<19 nM) and in the presence (~30 μM) of Caely Fig. . Basal G37. All parameters describing the phototransduction cascade were left unaltered, the only difference being the way the reaction describing the dynamic synthesis of cGMP by GC1 was implemented. In the present implementation, to account for specific contributions of GCAP variants to the cGMP synthesis in a mouse photoreceptor, each variant (GCAPi) was described to contribute by a specific fraction fi to the maximal activation (αmax) of GC1:A previously developed comprehensive biochemical model of phototransduction in mouse rods was used for numerical simulationsαmax, assumed to be the same for each variant, was fixed to 120 μM/s, as in the previous model, while IC50 and hc for WT-GCAP1 and GCAP2 were fixed to their experimentally measured values in mouse rod outer segments7. IC50 and hc for E111V-GCAP1 were set to their relative values (-fold variation) compared to the wild type, according to the parameters experimentally measured for the human orthologs (Table fi is a convenient way to account for the specific contribution of each GCAP variant to the overall regulation of GC1, with the assumption of a similar apparent affinity between GCAP variants and GC1, so that the variants may compete for the target based solely on their concentration/expression levels. This condition has been demonstrated to be realistic for E111V and WT human GCAP1, for which substantially unaltered EC50 values for human GC1 were measured10, as well as for murine GCAP1 and GCAP2, which showed almost identical EC50 values for murine GC1 in rod outer segment membranes7.The value for fi terms one can simulate a broad variety of experimental conditions; for example, the situation of a heterozygous mouse E111V/WT, corresponding to the human adCORD case, in which half of the GCAP1 molecules are WT and half carry the E111V mutations, in the presence of normal amounts of WT GCAP2 was modeled as follows:With the above assumptions, by tuning the GCAP1WT = 0; fGCAP1E111V = 0.5; fGCAP2 = 0.5. Finally, the case of 3-fold excess of WT-GCAP1 over a background of E111V-GCAP1 and endogenous GCAP2 was obtained by setting: fGCAP1WT = 0.375; fGCAP1E111V = 0.125; fGCAP2 = 0.5, while the same excess in the absence of endogenous GCAP2 was simulated by: fGCAP1WT = 0.75; fGCAP1E111V = 0.25; fGCAP2 = 0. All numerical simulations were performed in Matlab as explained in earlier works37.The case of homozygous mouse E111V/E111V in the presence of endogenous GCAP2 was simulated by setting fSupplemental Information


ATP2C1 alleles encoding the secretory pathway Ca2+/Mn2+-ATPase, hSPCA1. The disease almost exclusively affects epidermis, where it mainly results in acantholysis of the suprabasal layers. The etiology of the disease is complex and not well understood. We applied a yeast based complementation system to characterize fourteen disease-causing ATP2C1 missense mutations in presence or absence of wild type ATP2C1 or ATP2A2, encoding SERCA2. In our yeast model system, mutations in ATP2C1 affected Mn2+ transport more than Ca2+ transport as twelve out of fourteen mutations were unable to complement Mn2+ sensitivity while thirteen out of fourteen to some extent complemented the high Ca2+requirement. Nine out of fourteen mutations conferred a cold sensitive complementation capacity. In absence of a wild type ATP2C1 allele, twelve out of fourteen mutations induced an unfolded protein response indicating that in vivo folding of hSPCA1 is sensitive to disease causing amino acid substitutions and four of the fourteen mutations caused the hSPCA1 protein to accumulate in the vacuolar membrane. Co-expression of either wild type ATP2C1 or ATP2A2 prevented induction of the unfolded protein response and hSPCA1 mis-localization.Hailey-Hailey disease is an autosomal genetic disease caused by mutations in one of the two  ATP2C1 gene, encoding the Secretory Pathway Ca2+/Mn2+-ATPase Type 1 (hSPCA1) are known to cause HHD2 but the underlying mechanism of the disease is far from fully understood. The various model systems that have been used to study the molecular cause of HHD have revealed a disease with great complexity6.Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, dominantly inherited, autosomal skin-disorder, mainly characterized by loss of cohesion (acantholysis) between keratinocytes in the suprabasal layers of the skin. Mutations in the ATP2C1 have been deposited in the Human Gene Mutation Database Professional (www.hgmd.cf.ac.uk/ac/index.php), and more than 170 have been listed in recent publications8 only few have been functionally characterized9. The amino acid substitutions causing HHD do not cluster in specific parts of the hSPCA1 primary structure.Despite that currently more than 180 mutations in 10.hSPCA1 belongs to the family of P-type ATPases - a group of transmembrane proteins that uses the energy in ATP to transport one or more substances across the membrane, in a process involving transient phosphorylation of a conserved aspartate residue11. These hSPCA1 isoforms only differ in their carboxyl-termini. The shortest isoform, hSPCA1c has been shown to be nonfunctional, but the remaining hSPCA1a, -b and –d isoforms all have similar kinetic properties12. The precise functional differences among these three variants are still unclear but carboxyl termini of other P-type ATPases are known to play a regulatory role15.Four different splice variants of the human hSPCA1, named hSPCA1a-d have been experimentally verifiedtrans-Golgi16, where it transports Ca2+ and Mn2+ ions into the Golgi lumen17. ATP2C1 is ubiquitously expressed in mammalian cells but expression is particularly high in epidermal keratinocytes and varies among other human tissues19. hSPCA1 accumulation has been shown to be markedly reduced in keratinocytes from HHD patients16. While ATP2C1 mutations are disease causing, the complete etiology of the disease is apparently more complex, as only parts of the skin are affected and most patients are symptom-free for the first 2–4 decades of life. It is currently not known how mutations in ATP2C1 lead to the clinical phenotype and why mainly skin is affected. It is generally believed that the reduced ability to pump Ca2+ into Golgi, resulting from haploinsufficiency, is the main cause of HHD22. The suggestion that a perturbed cellular Ca2+ homeostasis is at the root of HHD is further supported by the fact that a Ca2+ has several important roles in the skin29.hSPCA1 localizes to Golgi membranes, particularly Saccharomyces cerevisiae hSPCA1 orthologue Pmr1 was the first secretory pathway Ca2+-ATPase to be identified and characterized32. Consequently, most of the knowledge we have on SPCA comes from studies of yeast Pmr1. Like its human counterpart, Pmr1 localizes mainly to Golgi but it is also present in low amounts in the ER membrane34. Like hSPCA1, Pmr1 transports Ca2+ and Mn2+ ions across the ER/Golgi membrane33, and one of its functions is to supply ER and Golgi resident enzymes such as glycosyltransferases, kinases and proteases with Mn2+ or Ca2+38. The requirement for Ca2+ ions in ER/Golgi may explain why pmr1Δ yeast cells are sensitive to very low extracellular Ca2+ concentrations40. pmr1Δ yeast cells are also hypersensitive to elevated extracellular Mn2+ concentrations42 as they cannot clear Mn2+ ions from the cytoplasm, which is essential, as an elevated Mn2+ concentration in the cytoplasm is toxic48.The 2+ requirement and the Mn2+ sensitivity of pmr1Δ yeast17. Yeast is a unique model organism for studying hSPCA1 mutations as it is currently the only background-free system that allows all endogenous intracellular Ca2+- and Mn2+ ATPases (PMR1 and PMC1) to be knocked out. Since yeast also lacks other secretory pathway Ca2+ pumps (i.e. SERCA-type Ca2+ ATPases) and Mn2+ pumps, potential interferences from these pumps are absent, too. Loss of both SPCA1 alleles in humans has only been described in very few cases50 and only as postzygotic, segmental loss of the healthy allele, with a more severe phenotype in the affected areas, indicating that SPCA1 is most likely essential. This is further corroborated by the fact that homozygous knock-out of SPCA1 or SERCA2 in mouse is lethal in the embryonal stage51. Taken together this indicates that a background-free mammalian model system may be unachievable.Human SPCA1 shows 49% homology with yeast Pmr1 and has previously been shown to fully complement the high Ca52, induces an evolutionary conserved and thoroughly investigated signal transduction pathway designated the unfolded protein response (UPR)53. In mammalian cells UPR can be induced by three different pathways, mediated by three different sensor proteins: PERK, ATF6 and IRE1. The IRE1 pathway is highly conserved among eukaryotes and also exists in yeast56. Thus, proteins that induce UPR in yeast are expected to induce UPR in mammalian cells, too. Accumulation of misfolded and unfolded proteins in the ER can lead to ER stress and prolonged or chronic ER stress can ultimately cause apoptosis57.Proper functionality of a protein is dependent on the specific three dimensional structure of the polypeptide chain. Interference with protein folding in the ER, e.g. due to amino acid substitutions, prevention of disulphide bond formation or inhibition of glycosylationSaccharomyces cerevisiae as model organism for heterologous expression of wild type (wt) and 14 missense variants of ATP2C1 identified in diagnosed HHD patients. We chose to investigate the shortest functional isoform, hSPCA1a, as this is the “canonical” isoform and because mutations affecting the function of the shortest form would most likely also affect the longer splice variants as only the C-termini differ among isoforms and no missense mutations affect this region7. We investigated how gene copy number affects the in-vivo Ca2+ and Mn2+ transport capacity of HHD mutants including its temperature dependency, intracellular localization and accumulation of hSPCA1a protein, in addition to ER-stress induced by expression of HHD variants. In addition we also investigated the effects of co-expressing wild type ATP2C1 or ATP2A2, encoding SERCA2.In the present study, we have used ATP2C1a gene dosage, temperature, protein accumulation, protein localization and interference with in vivo hSPCA1a protein folding may contribute to the etiology of HHD we selected fourteen disease-causing amino acid substitutions located in various domains of the hSPCA1a primary structure  or wt ATP2A2b (encoding hSERCA2b) and each of the investigated mutant alleles. The PAP4920 strain was selected because it is presently the only eukaryotic cell model without endogenous Ca2+ and Mn2+-ATPase activity and it is equipped with a chromosomally integrated unfolded protein response reporter58 for monitoring in vivo protein folding problems in the ER due to hSPCA1a expression.To address to what extent 2 concentration in the growth media from the standard 0.4 µM59 to 20 mM60 results in a concentration high enough to supply intracellular organelles with sufficient Ca2+ in the pmc1Δ pmr1Δ cnb1Δ yeast strain61. Requirement for a high extracellular Ca2+ concentration can be complemented by human wt ATP2C1a17. To obtain detailed information on how complementation depends on ATP2C1a gene dosage, yeast cells producing wt hSPCA1a or HHD variants from either a single cDNA copy (integrative) or approximately twenty copies  was significantly better than the empty vector but poorer than wt. HHD variant G789R stands out as it did not confer any BAPTA tolerance greater than that of the empty vector when expressed from a single cDNA copy and was only marginally better than empty vector when expressed from the multi copy plasmid. The BAPTA related phenotypes of all HHD variant are summarized in Table Data in Figs pmc1Δ pmr1Δ cnb1Δ yeast is very sensitive to Mn2+ as absence of Pmr1 abolishes removal of toxic amounts of Mn2+ accumulating in the cytosol17. To obtain detailed information on how ATP2C1a gene dosage influences complementation of the Mn2+ sensitive phenotype, yeast cells producing wt hSPCA1a or HHD variants from either a single cDNA copy or approximately twenty copies were grown in 96-well microplates at 30 °C in 12 different growth media 63. Since our yeast model system lacks PMR1, PMC1 and CNB1, Ca2+ may hyper-accumulate in the cytoplasm and generate a large concentration gradient across the membranes of ER/Golgi. Thus, even a poorly functioning Ca2+-ATPase may appear to have a significant Ca2+-transport activity. This could severely influence the observed difference in Ca2+ and Mn2+ transport of the investigated HHD variants   Fig. . The sam2+ or BAPTA at 30 °C from Fig. 2+-sensitive growth phenotype on media containing galactose and 0 mM BAPTA.When expressed from the replicative vector at 30 °C on agar plates, 10 of the 14 investigated HHD-mutants, including G309C, L318P, C411R, C490F, T570I, L584P, M641R, G645R, T709M and D742Y, displayed Ca2+-sensitivity in media containing galactose and 20 mM CaCl2 or galactose and 0 mM BAPTA. The observed growth inhibition is not simply caused by the presence of an excess amount of Ca2+ in the growth medium since all variants, including the empty vector (EV), sustained growth on agar plates containing glucose and 20 mM CaCl2 at 30 °C. The inhibition is also not caused by the combination of galactose and 20 mM CaCl2 since the negative control  could sustain growth at media containing galactose and 20 mM CaCl2. The inhibition is thus caused by expression of particular ATP2C1a alleles  and the level of expression of the HHD-alleles (as the growth inhibition was much more pronounced for the replicative variants) in combination with the particular concentration of Ca2+ in the medium.To simplify, the growth phenotypes observed on agar plates containing Ca2+ and no BAPTA was observed for the majority of the investigated HHD-alleles only HHD-variants L318P and C344Y showed growth inhibition at 0 mM BAPTA when expressed from the integrative vector , and expressed them from the replicative vector at 15 °C, 20 °C, 25 °C, 30 °C and 35 °C in the pmc1Δ pmr1Δ cnb1Δ strain PAP4920. Data in Supplementary Fig. 2+ ATPases at 30 °C and 35 °C while at 15 °C, 20 °C and 25 °C, it accumulated to 2–3 times the density observed for Pmc1 and Pmr1. HHD-variants A304T, G309C, L318P, I580V, G645R, T709M and D742Y protein accumulation was comparable to that of the wt hSPCA1a. Of these, G309C, G645R and T709M accumulated hSPCA1a protein in a more or less temperature insensitive manner. A304T and L318P protein accumulation peaked at 25 °C while both I580V and D742Y peaked at 30 °C. The remaining 7 HHD-variants all accumulated protein well below that observed for wt but on par with the Pmr1 orthologue. From the data summarized in Table In order to investigate how the combination of HHD mutation and temperature affects hSPCA1a protein accumulation, we C-terminally tagged wt- and the 14 different HHD hSPCA1a variants, as well as the yeast endogenous Capmc1Δ pmr1Δ cnb1Δ strain. To determine sub-cellular localization of wt and HHD hSPCA1a variants we expressed GFP tagged hSPCA1a variants and the endogenous Pmr1 and Pmc1 at 15 °C, 20 °C, 25 °C, 30 °C and 35 °C in the pmc1Δ pmr1Δ cnb1Δ strain. Live cell bioimaging of yeast cells producing GFP tagged Pmr1 or Pmc1   and therefore interactions with the binding site may affect Mn2+ more that Ca2+. It may seem counterintuitive that mutations preclude transport of the smaller Mn2+ ion, but not the larger Ca2+ ion. However, ion selectivity reflects the balance between the cost of ion dehydration and the benefits of interaction with the ion binding site69. As exemplified in K+ channels this results in a 10,000 times greater affinity for the larger K+ ion over the smaller Na+ ion. It has furthermore been shown that the selectivity of the K+ channel can be manipulated by adjusting the size of the ion-binding cavity69.Five of the investigated amino acid substitutions are located in the transmembrane part of hSPCA1 and four of these  are located in or close to the predicted ion-binding site created by amino acids in TM4, 5 and 6. G789R is located in TM7. None of the five mutations conferred Mn72. Apart from amino acid substitutions in the ATP or Mg2+ binding sites, substitutions in one part of the protein may thus affect ATPase activity by reducing or preventing structural transmission from the cytoplasmic domains to the ion binding site in the membrane. It is well established that shortcutting the essential communication between A, N, P and M domains by limited proteolysis prevents ATPase activity73. Because at least some Ca2+-ATPase activity is preserved in several of the analyzed HHD variants, communication between the cytoplasmic part and the cation sites cannot be completely blocked; it is more likely that communication is impaired in such a way that the geometry of the cation binding site in the trans-membrane domain is altered and affects the smaller Mn2+ ion more than the larger Ca2+ -ion.The reaction cycle of P-type ATPases involves large rearrangements and transmission of conformational changes initiated in the cytosolic A-, N- and P-domains and transduced to the membrane bound M-domain and vice-versa through long-range interactions between residues that are distantly remote in the primary structure74 either by compromising the folding pathway or the stability of the finally folded structure. The circumstance that yeast can grow at a wide temperature range offers a unique opportunity to demonstrate for the first time, that nine out of the fourteen HHD mutations analyzed in the present study exposed a cold sensitive phenotype as complementation was absent at the lower temperatures  By resulting in a non-functional or poorly functioning Ca2+-ATPase that is unable to counteract the constant Ca2+-leakage out of ER; 2) By resulting in an inactive, leaky protein that allows Ca2+ ions to more or less passively leak out of the ER; or 3) By misfolding.Despite their diverse phenotypes it is very interesting that homozygous expression of twelve out of the fourteen investigated HHD variants induced an unfolded protein response, because this may pave the way for a completely new way to target HHD. ER-stress has earlier been suggested to play an important role in the development of HHD due to the important role of Capmr1Δ yeast in low Ca2+ media, we supplement the growth medium with 20 mM CaCl2 as this fully restores growth and UPR to wt levels61. The normal feedback inhibition of HACS is also absent in our yeast strain. Addition of 20 mM CaCl2 to the growth medium thus creates a large enough Ca2+ gradient from the outside, through the cytoplasm and across the ER membrane to allow a sufficient amount of Ca2+ to enter the ER lumen, to allow growth and suppress UPR. It has previously been shown that mutations in ATP2A2 can result in leaky SERCA2 proteins82. Under normal physiological conditions Ca2+ would leak out of the ER since the Ca2+ concentration in the ER is generally much greater than that of the cytosol. Under our growth conditions, the electrochemical gradient supports Ca2+ influx into the ER, and Ca2+ leakage out of the ER thus seems unlikely. Misfolding itself could theoretically trigger UPR by bona-fide misfolding  or by sequestering BiP and thereby increasing Ca2+ leakage through translocons87. As leakage out of ER is unlikely, UPR in our experiments most likely reflect misfolding of the expressed HHD-hSPCA1a variants.Mutations resulting in poorly functioning or non-functioning hSPCA1a are expected to behave in a similar manner to the empty vector Fig.  and the Amino acids 723 to 729 , expose the most probable BiP binding site  and both single mutants grow well at 100 mM CaCl240. pmc1Δ pmr1Δ cnb1Δ yeast have previously been shown to grow well at 20 mM CaCl260 and up to 200 mM CaCl292. The negative control (empty vector) did not display Ca2+ sensitivity in our experiments. Addition of 20 mM CaCl2 to the medium did not have an effect in the presence of glucose (i.e. when ATP2C1 is not expressed). Interestingly, only the replicative variants displayed Ca2+ sensitivity on media containing galactose and 20 mM CaCl2, indicating that it is the combination of high expression and the specific HHD variant that is responsible for this phenotype, as neither expression of wt nor the negative control conferred any Ca2+ sensitivity. We did not observe a clear correlation between Ca2+ sensitivity and other phenotypes investigated in our study and it is thus difficult to draw any conclusions on what may cause the observed Ca2+ sensitivity. However, keratinocytes defective in hSPCA1 display an inability to properly accumulate Ca2+ upon increases in extracellular Ca2+16, and a similar observation has also been made with yeast defective in Pmr190. Ca2+ sensitivity has even been suggested to be the most relevant phenotype for yeast pmr1 mutants in resembling keratinocyte pathology29.An interesting phenotype displayed by several of the investigated mutations is sensitivity to Ca2+ sensitivity (12 out of 14) are the two most dominating phenotypes. This may indicate that HHD disease is a conformational disease and Mn2+ homeostasis may be more affected than Ca2+ homeostasis. A new approach towards treatment of HHD may thus focus on development of chemical chaperones to combat the hSPCA1 folding problems we have demonstrated.In conclusion, results obtained in our yeast model system demonstrate that while the phenotypes exposed by our panel of HHD mutants are very diverse, UPR induction (12 out of 14) and Mnade2Δ1 can1-100 his3Δ 11.15 leu2Δ 3.112 trp1Δ 1 ura3Δ 1 pmc1::TRP1 pmr1::HIS3 cnb1::LEU2)60 was a kind gift from Kyle Cunningham, John Hopkins University, USA. Strain PAP4920 was generated by insertion of an unfolded protein response reporter, UPR-lacZ, into the ade2Δ1 locus of K616. Strain PAP9588 was constructed by inserting a nourseothricin resistance gene from plasmid pAG2586 into the TRP1 gene of PAP4920 by homologous recombination. The UPR-lacZ reporter is identical to that previously described58 except that the ADE2 marker substituted for the LYS2 marker. PAP1500 (α ura3–52 trp1:: GAL10-GAL4 lys2–801 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6 R can1 GAL) has been described previously65. Yeast strain Y04534  was purchased from Euroscarf.The yeast strain K616  into similarly digested pEMBLyex493. The integrative Gateway® compatible expression vector pPAP5480 was constructed by relegation of MfeI digested pPAP4997. The replicative Gateway® compatible expression plasmids pPAP7177 and pPAP8754 were constructed by replacing the URA3 gene in pPA4997 with the ADE2 gene or the TRP1 gene, respectively.The integrative Gateway® compatible expression vector pPAP7010 was generated by inserting an ApaI-CYC-GAL-promoter- attR1-ccdB-CmR-attR2-BglII PCR fragment amplified from pPAP4997 into ApaI, BamHI digested pRS40294. Plasmids expressing either wt or amino acid substituted human hSPCA1a were generated by overlap extension PCR95 using an ATP2C1a cDNA clone obtained from Invitrogen, USA. Final PCR products were transferred by Gateway® Technology to pDONOR221 . After confirmation of the correct nucleotide sequence, all ATP2C1 alleles were transferred to pPAP4997 and pPAP5480 by LR-recombination. wt ATP2C1 was furthermore LR recombined into pPAP7010, pPAP7177 and pPAP8754. C-terminal in frame fusions to yEGFP96 was created by in vivo homologous recombination in yeast between SacI or HindIII digested ATP2C1 expression plasmids and yEGFP PCR fragments. C-terminal tagging of wt ATP2C1 with BFP was generated by in vivo homologous recombination in yeast between SalI, HindIII digested pPAP8754, a wt ATP2C1 PCR fragment and a codon optimized BFP  PCR fragment.The replicative Gateway® compatible expression vector pPAP4997 was generated by insertion of a 2. Cultures maintained in the exponential growth phase were harvested, washed five times in sterile 18 mΩ water and subsequently re-suspended in 18 mΩ water to an OD450 = 0.5. 7 µl of this cell-suspension was spotted on minimal plates containing galactose as sole carbon source (SG plates) and either 20 mM CaCl2, BAPTA or MnSO4. Sterile filtered CaCl2 (from 1 M stock in dH2O), BAPTA  or MnSO4 (from 100 mM stock in TES/KOH pH = 6) was added to the media to obtain the required concentrations. Plates were incubated at 15 °C, 20 °C, 25 °C, 30 °C or 35 °C, inspected daily and photographed.Yeast cells inoculated from −80 °C stock were grown at 30 °C in liquid synthetic minimal medium containing 2% glucose as sole carbon source and 20 mM CaCl2, BAPTA or MnSO4  to the desired final concentrations. 20 µl of a cell suspension with OD450 = 0.5 in dH2O was added to each well. Plates were incubated at the desired temperature and OD450 of the cultures measured in a plate-reader  at appropriate time intervals. BAPTA ethane-N,N,N’,N’-tetraacetic Acid) was purchased as tetrasodium salt from Merck (cat.no.: 196418).Yeast cells were cultured from −80 °C stock as above. Complementation assays were performed in clear 96-well plates (well capacity: 400 µl). Each well contained 200 µl yeast culture. Medium was made from 100 µl 2x SG medium and 80 µl of CaCl2 and washed five times in 18 mΩ H2O. Each culture was used to inoculate shake flasks containing 50 ml of galactose minimal medium and either 20 mM CaCl2, 0 mM CaCl2 or 0.06 mM BAPTA to OD450 = 0.05. Shake flask were inoculated at 15 °C, 20 °C, 25 °C, 30 °C or 35 °C. Cultures were harvested when OD450 reached 1 to 1.5, washed in 18 mΩ H2O and immediately frozen at −80 °C. Crude yeast membranes and cytosolic fractions were isolated as previously described65.Yeast cells were grown in SD medium supplemented with 20 mM CaCl97 according to the manufacturer’s specifications . β-galactosidase activities were determined in purified cytosolic preparations as described previously58.Protein concentrations in cytosolic- and membrane preparations were determined by the BCA method98 were visualized at 1,000 x magnification with a Nikon Eclipse E600 fluorescence microscope equipped with an Optronics Magnafire model S99802 camera attached or a Leica SP5-X confocal microscope.C-terminally GFP/BFP tagged hSPCA1a proteins, N-terminally BFP tagged SERCA2b and FM4-64 stained cellsATP2C1a-GFP protein in crude yeast membranes was quantified using a standard curve made from purified GFP protein as described previously99.The density of ATP2C1was performed according to Blond-Elguindi et al.100. ER resident parts were identified by the TMHMM software101 while potential interactions between transmembrane helixes were predicted by the TMHIT program102.Prediction of BIP binding sites in ER resident parts of Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism


The computational model presented here combines cellular pathways, fluid dynamics, and physiologically-realistic vessel geometries as observed in the human vasculature. The coupled cells model includes endothelial cells (ECs) and smooth muscle cells (SMCs) with ion dynamics, hetero and homotypic coupling, as well as electro-diffusive coupling. Three arterial bifurcation surface models were used in the coupled cells simulations. All three simulations showed propagating waves of Ca2+ in both the SMC and EC layers, following the introduction of a luminal agonist, in this case ATP. Immediately following the introduction of ATP concentration Ca2+ waves propagate from the area of high ATP toward the areas of low ATP concentration, forming complex patterns where waves interact with eachother, collide and fade. These dynamic phenomena are repeated with a series of waves of slower velocity. The underlying motivation of this research was to examine the macro-scale phenomena, given that the characteristic length scales of atherosclerotic plaques are much larger than a single cell. The micro-scale dynamics were modeled on macro-scale arterial bifurcation surfaces containing over one million cells. The results of the simulations presented here suggest that susceptibility to atherosclerosis development depends on the bifurcation angulation. In conjunction with findings reported in the literature, the simulation results demonstrate that arterial bifurcations containing wider angles have a more prominent influence on the coupled cells pathways associated with the development of atherosclerosis, by means of disturbed flow and lower SMC Ca2+ concentrations. The discussion of the results considers the findings of this research within the context of the potential link between information transport through frequency encoding of Ca2+ wave dynamics and development of atheroprone conditions.This work investigates the effect of arterial bifurcation angulation on atherosclerosis development through  Over the past 30 years, since the pioneering research of Caro et al. , a substMultiple studies agree that disturbed vascular flow is the potential trigger for both endothelial and smooth muscle cell dysfunction in human vasculature, and lead to the activation of pro-inflammatory phenotype and coronary plaque formation Libby, . SimilarFurther, it has been shown that the localization of plaque formation is associated with properties of arterial geometries especially in or close to the coronary and carotid bifurcations  is the mediator for protein phosphorylation the work of Goldbeter et al.  pathway has been shown to be a crucial signaling mechanism in foam cell formation, endothelial cell (EC) activation, and vascular smooth muscle cell (SMC) proliferation , taken from Bennett et al. /2, which is the average concentration of ion ϕ between two cells a and b; Asegment is the interface area of neighboring cells; and δx is the distance between cells. The concentration gradient here is only relevant for Ca2+ in our model, as intracellular concentrations of all other ions were assumed to remain constant on the considered time scale. The values of these constant concentrations are taken from Kapela et al.  were added to the hetero and homocellular coupling terms for membrane potential. The definitions, values, and dimensions of these parameters are provided in the 3 coupling due to its zero valence is coupled using Fickian diffusion.Here a et al. . The ionThe macroscopic mean conductance G in membrane potential coupling has been estimated by Van Rijen et al.  to be 30n et al.  yield a n et al. . Therefon et al. .−1.The membrane capacitance value between ECs is of the order of 30 pF as given by Schuster et al.  and for 2+ and IP3 transfer is not available. The work of Koenigsberger et al.  may decrease Ca2+ levels, but experimental evidence on this pathway is still controversial.Fast  with a possible reduction in [Ca2+] oscillations. Potentially, this may have the effect of inhibiting the MAPK pathway, and possibly inhibiting the initiation of plaque growth. However, the model of Hadfield et al. -enabled parallel environment. A periodic boundary condition was imposed along the longitudinal edge of each 2D grid. In this case, the periodic boundary condition refers to the communication-specific mapping of MPI tasks/domains. Each domain included 208 ECs and 80 SMCs, coming to 288 cells per quadrilateral, with a total of 1,175 million cells, spread across 4080 quadrilateral domains. The parallel implementation of the coupled cells simulations presented here is based on the work of Zakkaroff et al. .The system of ODEs simulating the time-dependent transfer of ions and membrane potential within each quadrilateral domain was solved at a specified time-step. For the simulations presented here the inter-domain communication interval was set to 0.01 s. This interval was chosen on the basis of a number of tests to maintain the balance between MPI communication and computation times. At regular time intervals the state variables from the cells along the edges of the quadrilateral domains were passed to the adjacent cells in the neighboring domains. The inter-domain communication was implemented through the MPI library, the solution of which follows the mesh ghost cell communication pattern recommended for discretised domain decomposition , and the SMC [Ca2+] values are again near-constant, but lower, on average 0.4 μM (data not shown).Figure 2+ in both the SMC and EC layers following the introduction of a luminal agonist, in this case ATP. The agonist binds to endothelial receptors, induces the production of IP3, and consequently the release of Ca2+ in both ECs and SMCs. Both ECs and SMCs are excitable and their Ca2+ concentrations can oscillate under certain conditions  pathways in a variety of cells have been shown to be important for the development of atherosclerosis.Research by Goldbeter and Koshland  and Gold2+ dynamics alone, aligned with variation in geometry and luminal wall agonist concentration, we conclude that initiation of atherosclerotic plaque may not be due to frequency encoded Ca2+ phosphorylation of the protein alone. However, combinations of more complex cellular models and the introduction of other pathways such as NO, along with the use of numerical simulations could provide further insight into the relationship of vascular geometry and atherosclerotic plaque formation.It emerges that no single marker exists for plaque formation and that a number of conditions have to be present in order to initiate atherosclerotic conditions. On the basis of Ca3 mediated Ca2+ pathway, and gap junction currents that replace simple linear gradients for membrane potential and Fickian diffusion of ions. The premise of this work was such that variations in the frequency and dynamics of the Ca2+ waves in areas of low WSS have the potential to influence the kinase reactions of proteins under the assumption that Ca2+ encodes for high sensitivity phosphorylation and possibly provides a marker for atherogenesis.This research examined single-cell dynamics on a macro-scale, and the presented model was compared with other mathematical models of cellular physiology in the related literature. The underlying coupled cells model was extended to include a detailed IP2+ concentrations were observed in areas under the lateral surface of bifurcations, in SMCs. The SMC Ca2+ dynamics in these areas were difficult to analyse at any given time point: waves propagated in a slow non-uniform manner, and over significantly shorter distances. Post-processing, in the form of time-averaging, was applied to gain insight into the emergent properties of these dynamics. This showed significantly lower Ca2+ concentrations over time at the lateral bifurcation seams compared to the other surface regions of the arterial segments. Ca2+ concentration frequencies and average values in the lateral domain for SMCs were relatively small. In contrast EC Ca2+ dynamics showed that average values were high in both apical and lateral regions. Since experiments have shown that plaque growth is rare in the apical region this would indicate that frequency encoding would not be a prominent mechanism in the very early stages of plaque growth.The macro-scale simulations in this work demonstrated oscillatory behavior in SMC and EC species' concentrations. Moreover, significantly lower Ca2+ concentrations. The overal outcome of this work suggests that although Ca2+ dynamics are of considerable importance in cellular functions, other related pathways may combine in a non-linear fashion to produce an environment conducive to plaque formation.In conjunction with findings reported in the literature, the results of this work suggest that arterial bifurcations containing wider angles may indeed influence the development of atherosclerosis, by means of disturbed flow and, consequently, lower SMC CaThis research provides a flexible simulation platform that allows for the investigation of cellular dynamics with geometrically varying, multi-scale arterial sections. The potential influence of bifurcation angulation on atherogenesis has been studied, and the investigation of many other phenomena is now possible. Finally, all additions and modifications made to this platform were designed and implemented to promote scalability and further model development. Future simulations of arterial physiology, including further detailed cellular chemistry and surface-meshes that span the entire coronary tree, should benefit from these extensions.in-silico experiments; however, we hope that future research in physiology may help improve the homogenization of experimental findings for computational modeling.It is important to note that biophysical values and coefficients used in the presented model were collected from different configurations of experiments. For example, the coefficients of electro-diffusive coupling can vary widely depending on experimental setup. These considerations present a potential limitation for For a given mesh, the ECs and SMCs on each quadrilateral were aligned orthogonally with respect to each other, where ECs align axially with the flow and the SMCs align circumferentially. These arrangements were generated such that they communicate with four neighboring cells homocellularly, when in fact an EC and SMC can typically communicate with up to six neighboring ECs and SMCs, respectively  in the EC, was not included in this model. This pathway describes the production of NO as a result of agonistic molecules within the lumen binding to EC receptors. NO has known atheroprotective properties, such as inhibiting the production of adhesion molecules that encourage the recruitment of monocytes. Following the implementation of this pathway in macro-scale simulations, it will be possible to examine NO dynamics across large sections of ECs, and, possibly, provide further insight into the process of atherosclerotic plaque development. Similarly, the introduction of stretch-activated channels and vasculature motion in the model may improve the fidelity of future simulation results.Many geometric variations were considered in a study conducted by Lee et al. , among w2+ dynamics simulations in order to model both healthy and pathological conditions in coupled cells behavior. These coupling cases were not the focus of the research presented here—the particular coupling cases modeling unhealthy cellular behavior already assume mid-to-late stages of atheroma development. However, future work should include the creation of new coupling cases that reflect initial stages of disease, before plaque formation is observable. Further, these coupling cases should vary both temporally and spatially as noted above due to the variation in EC alignment. Similarly, in their discussion of Ca2+ dynamics in the vascular endothelium, Taylor and Francis (2+ changes and instead focus on spatially and temporally relevant aspects of real-time signaling.Shaikh et al.  introduc Francis  stated t2+ concentrations, would allow us to simulate the progression of vascular disease in relatively short simulations.The results of our simulations show abnormal cellular dynamics at specific locations on arterial geometries; therefore different coupling parameters should be employed at these locations to reflect potentially emergent dysfunctional cellular communication, but there seems to be little evidence to indicate the coupling parameters variation with respect to WSS. This is particularly important, given that simulations presented here span physiological time periods of minutes, while atherosclerotic plaques develop over time scales of months. Modification of localized communication channels as a function of atypical cellular dynamics, such as significantly lower Cahttps://github.com/BlueFern/CoupledCells/releases/tag/v-4.0.The computational model and required input files for this study can be found in the Coupled Cells Github release: All authors contributed equally to the direction and ideas of the presented work. SD and CZ generated the new surface meshes, extended the physiological model, and ran parallel simulations. SM produced the input ATP maps using computational fluid dynamics methods. SD, CZ, and TD developed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIβ (PI-4K-β) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.The EF-hand calcium (Ca Changes in intracellular Ca2+ are transduced by multiple proteins, with a key role of a large family of EF-hand Ca2+-binding proteins that act as Ca2+ sensors or Ca2+ buffers. Ca2+-buffer proteins chelate Ca2+ and often terminate Ca2+ signals  is the mammalian homolog of the Drosophila frequenin protein, which belongs to the larger NCS protein family that includes NCS-1, visinin-like proteins, recoverin, guanylate cyclase-activating proteins, and potassium channel-interacting proteins (KChIPs). The structure of NCS-1 is shown in the 2+-binding protein containing 4 EF-hand motifs; of these, 3 (EF2-4) bind to Ca2+. Unlike ubiquitously expressed calmodulin, NCS-1 is predominantly expressed in the brain and cardiac tissues, suggesting its specialized roles in these tissues. The Ca2+-binding affinity of NCS-1 is significantly higher than that of calmodulin . Although both Ca2+-binding proteins can operate within the physiological range of Ca2+ levels (~100 nM to ~1–5 μM), the above data suggest that NCS-1 may be more sensitive to small changes in intracellular Ca2+.Intracellular calcium , voltage- and ligand-gated ion channels, and interleukin-1 receptor accessory protein like-1 (IL1RAPL1). Here, we summarize the current understanding of NCS-1 regulation of some of these target proteins and how it affects brain and cardiac functions . Even th2+-bound yeast NCS-1 (Ncs1) in complex with an N-terminal yeast PI-4-K (Pik1) fragment  catalyzes the synthesis of phosphatidylinositol 4-phosphate, which is a late limiting step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of many cellular functions including exocytosis. A yeast homolog of NCS-1 and PI-4-K interact, and NCS-1-induced activation of PI-4-K is required for yeast survival -induced enhancement of neurotransmitter release in motoneurons , including NCS-1, calcyon, and DARPP-32. NCS-1 directly interacts with the D2 dopamine receptor, inhibits D2 receptor phosphorylation, and reduces the agonist-mediated internalization of the receptor , a key regulator of memory function, and dopamine were decreased. Furthermore, phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α), which regulates long-term potentiation, and BDNF levels were decreased, suggesting that CaMKII-α signaling that increases BDNF production is at least partly involved in NCS-1-mediated learning and memory function.Several studies have demonstrated that NCS-1 modulates learning and memory. For example, deletion or reduction of NCS-1 resulted in dysfunction of learning and memory in 2+ signaling via inositol 1,4,5-trisphosphate receptors (InsP3Rs) regulates cellular function and is involved in pathogenesis  Ca2+ content were significantly lower in Ncs1−/− myocytes at the neonatal stage than in wild-type cells. Mechanistically, the interaction of NCS-1 with InsP3R increases InsP3R-dependent Ca2+ signaling, followed by the activation of CaMKII-dependent pathways, and promotes SR Ca2+ pump via the phosphorylation of phospholamban (PLB), which ultimately induce increase in the SR Ca2+ content and global Ca2+ transient, thus cardiomyocyte contraction  coupling in the postnatal stages. NCS-1 may act as one of these missing factors. Numbers of molecules which levels are high at the immature stage are often up-regulated in the disease conditions, such as cardiac hypertrophy. NCS-1 is also highly expressed during the early stages of hypertrophy in the adult heart and promotes the progression of hypertrophy, at least in part, through InsP3R activation . Recent evidence suggests that gene transcription is regulated by nuclear Ca2+ signals. However, the mechanisms underlying nuclear Ca2+ regulation and its relationship to cytoplasmic Ca2+ regulation have not been completely solved. Using a subcellular-specific, fluorescent protein-based Ca2+ indicator GECO  in neonatal mouse ventricular myocytes; and (2) receptor stimulation-elicited nuclear Ca2+ transients were mainly mediated by InsP3Rs. Furthermore, based on the evidence that IGF-1-elicited nuclear Ca2+ transient was significantly diminished in Ncs1−/− cardiomyocytes, NCS-1 is involved in the receptor stimulation-induced nuclear Ca2+ regulation through interaction with InsP3Rs , suggesting a general role of Ca2+ influx via the channel that binds to NCS-1 and consequently regulates channel functions and/or downstream Ca2+-dependent signaling, which affect various neuronal and cardiac functions. Furthermore, many established roles of NCS-1 are related to protective responses of cells against exogenous stress that leads to mild increases in cytosolic Ca2+. This suggest that intracellular Ca2+ as a determinant of cell survival and cell death, and Ca2+-sensor proteins, such as NCS-1, may serve as a switch to proceed the signal. We believe that this review provides intriguing observations and compels researchers to conduct detailed investigations and extend their studies on NCS-1 and its important regulatory proteins.Recently, studies have elucidated new roles of NCS-1 in physiology and pathophysiology. In this review, we have mainly focused on NCS-1 in the neuronal system and heart. Our particular interest is the emerging theme that NCS-1 directly regulates the function of several ion channels that permeate CaTN conducted most part of research, wrote, organized, and finalized the article. SN did some experiments and wrote some part of the article. SW contributed to the discussion on all part of the study and wrote some part of the manuscript. SN and SW wrote and edited some parts of the article.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.


Depolarization accompanies neuronal firing and elevation of Ca2+ triggers neurotransmitter release from synaptic vesicles. For synchronization of efficient neurotransmitter release, synaptic vesicles are targeted by presynaptic Ca2+ channels forming a large signaling complex in the active zone. The presynaptic CaV2 channel gene family  encode the pore-forming α1 subunit. The cytoplasmic regions are responsible for channel modulation by interacting with regulatory proteins. This article overviews modulation of the activity of CaV2.1 and CaV2.2 channels in the control of synaptic strength and presynaptic plasticity.Presynaptic Ca Multiple mechanisms regulate the function of presynaptic Ca2+ channels ,65V2.1 a SNAP-25 ,67, suggV2.1 and CaV2.2 channels regulate channel activity . Syntaxin-1A or SNAP-25 shifts the voltage dependence of inactivation toward more negative membrane potentials and reduces the availability of the channels to open  an an65] an(CaMKII) . The negrylation . Thus, p2+ elevation regulates CaV2.1 channels activity by its binding to CaM [2+-binding proteins, CaBP1, VILIP-2 [V2.1 channel proteins consist of a pore-forming α1 subunit associated with β, and possibly α2δ subunits  and I–II loop  evoked by various patterns of presynaptic APs firing in the presence of the blocker of endogenous CaV2.2 channels [At most fast synapse in the central nervous system, Ca synapse ,108, is channels ,110. TheG neuron . Sectionchannels .2+ channels has a powerful influence on synaptic transmission [V2.1 channel mediates transmitter release from the transfected SCG neurons [V2.1 channel interacting with Ca2+ bound CaM  and facilitation (PPF)  a. PPD wiefficacy b. The tiV2.1 channel. Mutation of the IQ-like motif potentiated reduction of the release efficacy, whereas the deletion of the CBD increased the release efficacy  that possess four EF-hand Ca2+-binding motifs. CaBP-1, VILIP-2, and NCS-1 bind to the same site as CaM, and modulate CaV2.1 channel activity. CaBP1, highly expressed in the brain and retina [V2.1 channels, binding to the CBD [2+-dependent facilitation of CaV2.1 channels but inhibits Ca2+-dependent inactivation of CaV2.1 channels, binding to both IQ-like motif and CBD [2+-dependent inactivation of P/Q-type Ca2+ currents through interaction with the IQ-like motif and CBD without affecting peak current or activation kinetics [CaBP1, VILIP-2, and NCS-1 are members of a subfamily of neuron-specific Cad retina , causes  the CBD . VILIP-2 the CBD , increas and CBD . NCS-1, kinetics .V2.1 channels with binding residual Ca2+ [V2.1 channels, significantly reduced the synaptic facilitation and enhanced the synaptic depression , and PPF with intermediate interval (20–100 ms)  a. In consynapses .2+/CaM-dependent regulator of postsynaptic response [V2.1 channels upstream of the IQ-like motif, and enhances the activity by slowing inactivation and positively shifting the voltage dependence of inactivation [V2.1 channels of SCG neurons prevented both PPD and PPF, suggesting that binding of CaMKII to CaV2.1 channels is required for the expression of this regulatory effect. Similarly, the expression of the brain-specific CaMKII inhibitor CaMKIIN [V2.1 channels [V2.1 channels by bound CaMKII controls the activity of those channels that have the effector of the Ca2+ signal  in position to bind the entering Ca2+ and respond to it [2+ signal for initiation of SVs exocytosis increasing the activity of the CaV2.1 channels by the formation of a complete SNAREs complex with synaptotagmin and RIM bound [2+ entry through those Ca2+ channels whose effectors are bound and ready to respond.CaMKII is the most prominent Caresponse ,120,121 response ,124,125.response , which dresponse , binds ttivation . The deptivation . The pre CaMKIIN , which pchannels , also prnd to it . SNARE pIM bound ,70. ThisV2.1 channels also binds to synapsin-1, a phosphoprotein of the SVs, increases its phosphorylation and induces oligomers of synapsin-1 [V2.1 and synapsin-1 bound to CaMKII would modulate the dynamics of SV function in AZs containing these proteins [Furthermore, autophosphorylated CaMKII bound to Canapsin-1 . Synapsinapsin-1 . The phonapsin-1 ,123. Forproteins .2+ channels by Ca2+ bound CaM and nCaS and causes facilitation and inactivation of neurotransmitter release. The differential expression of these Ca2+-dependent regulatory proteins may provide a means of cell-type-specific regulation of presynaptic Ca2+ channels and short-term synaptic plasticity. The short-term plasticity of neurotransmitter release shapes the postsynaptic response to bursts of impulses and is crucial for the fine-grained encoding of information in the nervous system [Neuronal firing regulates presynaptic Cas system ,130.2+ current by voltage-clamp methods, suggests that neuronal firing controls P/Q- and N-type currents to modulate differentially synaptic transmission. Presynaptic Ca2+ current consists of a combination of P/Q- and N-type currents in young mice and shows activity-dependent facilitation that predicts the amount of synaptic facilitation according to the power law [V2.1 knockout lost both facilitation of the presynaptic Ca2+ current and synaptic facilitation [2+ currents are less efficient in mediating synaptic transmission and do not support facilitation of synaptic transmission, but they are more sensitive to modulation by G protein-coupled receptors [V2.1 channels are responsible for neuronal activity-dependent synaptic facilitation, while CaV2.2 channels have strong G protein regulation.The Calyx of Held, the large presynaptic terminal enabling to record directly presynaptic Caower law ,132. CaVlitation ,131,132.eceptors . These r2+. The optical measurement of presynaptic Ca2+ transients with the induction of PTP in the calyx of Held showed an increase in the Ca2+ influx to the extent that predicted PTP when the power law of neurotransmission was applied, and the Ca2+ transient decayed with a time course of the decay of PTP [V2.1-transfected SCG neurons, PTP was not significantly affected by mutations at the IQ-like motif [2+ entry through CaV2.1 channels by CaM- and nCaS-binding in an activity-dependent manner, which in turn facilitates neurotransmitter release. It is likely that facilitation of presynaptic Ca2+ currents may contribute to short-term facilitation [Presynaptic short APs bursts generate augmentation and longer APs bursts generate post-tetanic potentiation (PTP) relying on residual Cay of PTP . In CaV2ke motif . In contlitation ,132, andlitation .Vβ subunits has a strong influence on synaptic facilitation in hippocampal synapses through their effects on Ca2+ channel function [2+ channels within the presynaptic terminal in a subunit-specific manner and may be involved in the organization of the Ca2+ channel relative to the release machinery [The expression of Cafunction . Cavβ2 aachinery .V2.1 channels at the IQ-like motif in hippocampal neurons confirmed the mechanism of short-term synaptic facilitation dependent nCaS regulation of CaV2.1 channels with brief and local Ca2+ elevation [V2.1 channel mutation at the IQ-like motif are associated with pronounced deficits in spatial learning and memory in context-dependent fear conditioning and in the Barnes circular maze. Thus, regulation of CaV2.1 channels by CaM and nCaS is required for not only presynaptic facilitation but also induction of postsynaptic long-term potentiation, and spatial learning and memory [The mutation of Calevation . In addid memory .2+ current in the AZ. Presynaptic loading of peptides that disrupt CaM interactions reduced both Ca2+-dependent inactivation of the P/Q-type Ca2+ current and PPD [2+-dependent inactivation of the presynaptic Ca2+ current, rather than SVs depletion, causes rapid synaptic depression for stimuli ranging from 2 to 30 Hz [At the calyx of Held, presynaptic stimulation at 100 Hz induces robust synaptic depression . Synapti and PPD . The Ca2to 30 Hz ,88.V2.1 channels lacking the CBD, a mutation reducing Ca2+-dependent inactivation in heterologous expression systems [2+-dependent facilitation of P/Q-type Ca2+ current, induced PPD, and synaptic depression during APs burst. However, the synaptic depression was absent in the presynaptic neuron coexpressed with CaBP1 and CaV2.1 channels lacking the CBD [V2.1 channel bound with CaM or CaBP1. During APs burst at 30 Hz and 40 Hz, a slower phase of synaptic depression is likely caused by SVs depletion.The transfection of SCG neurons with Ca systems ,81, bloc systems . CaBP1 e the CBD . These rV2.1-transfected SCG neurons suggest a conserved mechanism for generating rapid synaptic depression evoked by physiological rate and duration (at 40 Hz for 1 s) of APs bursts in multiple synapses where neuronal activity elevates presynaptic Ca2+ transient, and such a Ca2+ rise dependent binding of nCaS to CaV2 channels inactivates presynaptic Ca2+ channels. Studies of β subunits within cultured hippocampal neurons also support an important role for CaV2 channels modulation in synaptic plasticity: the overexpression of CaVβ4 favors facilitation whereas the overexpression of CaVβ2 favors depression [Data from the calyx of Held and Capression .V2.1 channels enhances their functional activity by inhibiting their inactivation [V2.1 channels or SCG neurons transfected the brain-specific CaMKII inhibitor CaMKIIN [V2.1 channels [V2.1 channel is required for both up-regulation of channel activity in presynaptic facilitation and for Ca2+-independent activation of CaMKII by CaV2.1, and one or both of these effects is necessary for normal short-term synaptic plasticity.The binding of CaMKII to Cativation  and enhativation . SCG neu CaMKIIN  which prchannels  prevente2+ sensors to mediate the fast synchronous transmitter release as discussed above [2+ via its C2A domain [Synaptotagmin-1, 2, and 9 serve as Caed above ,73,74. IA domain  is not rA domain . SynaptoA domain . SynaptoA domain  that may2+ buffers such as parvalbumin, calbindin, and related Ca2+-binding proteins control Ca2+ homeostasis [2+ buffer parvalbumin [2+ buffer calbindin [In the presynaptic terminal Caeostasis  and synaeostasis ,141,142.valbumin  controlsvalbumin . In contalbindin  alters salbindin .2+ channel modulation, synaptotagmin-7 action and Ca2+ buffering, activated by Ca2+ elevation with neuronal firing. Ca2+ channel modulation with CaM and nCaS is a response to millisecond Ca2+ dynamics. The slower synaptotagmin-7 action integrates local and global Ca2+ entry, and Ca2+ buffering may control the spread Ca2+ accumulation [Short-term plasticity may be a combination of the three molecular mechanisms, Camulation .2+ binding proteins triggers SVs exocytosis and regulate the probability. Thus, modulation of presynaptic Ca2+ channels has a powerful influence on synaptic transmission. At the pre-firing state, Ca2+ channels activity is inhibited by interaction with AZ proteins. AP firing relieves the inhibition by switching to interact with SNAREs and synaptotagmin, the effectors for Ca2+-dependent exocytosis. During and post firing, the activity of the CaV2.1 channel is regulated by interaction with CaM and nCaS dependent on individual speed and affinity of binding to residual Ca2+. Interacting with CaMKII, the CaV2.1 channel increases the binding to CaM and nCaS and their interaction causes short-term facilitation and depression of synaptic transmission. Fine-tuning the function and regulatory properties of presynaptic P/Q-type Ca2+ currents allow a greater range of input-output relationships and short-term plasticity. In contrast, tonic inhibition of N-type Ca2+ currents is activated by G-protein coupled-autoreceptors and retrograde signaling receptors.In response to presynaptic AP firing, Ca


To better understand how chloride transport affects lysosomal ion homeostasis and how its disruption impinges on lysosomal function, we developed a mathematical model of lysosomal ion homeostasis including Ca2+ dynamics. The model recapitulates known biophysical properties of ClC-7 and enables the investigation of its differential activation kinetics on lysosomal ion homeostasis. We show that normal functioning of ClC-7 supports the acidification process, is associated with increased luminal concentrations of sodium, potassium, and chloride, and leads to a higher Ca2+ uptake and release. Our model highlights the role of ClC-7 in lysosomal acidification and shows the existence of differential Ca2+ dynamics upon perturbations of Cl−/H+ exchange and its activation kinetics, with possible pathological consequences.The establishment and maintenance of ion gradients between the interior of lysosomes and the cytosol are crucial for numerous cellular and organismal functions. Numerous ion transport proteins ensure the required variation in luminal concentrations of the different ions along the endocytic pathway to fit the needs of the organelles. Failures in keeping proper ion homeostasis have pathological consequences. Accordingly, several human diseases are caused by the dysfunction of ion transporters. These include osteopetrosis, caused by the dysfunction of Cl Lysosomes are membrane-enclosed organelles of eukaryotic cells characterized by an acidic pH, an enrichment in hydrolytic enzymes and a specific composition of membrane proteins ,2. These+), 30–60 mM of H+ has to be pumped into the lumen in order to decrease the pH by one unit  of approximately 100 nM e and [Cl−]i are the cytosolic and luminal chloride concentration modified by a Boltzmann factor, respectively (ko (JClC-7ko) with Equation (3) by setting NClC-7 = 0. The initial luminal concentrations of K+, Na+, and Cl− concentrations were set to 50 mM, 20 mM, and 1 mM, respectively, as reported by Steinberg et al. e ectively . We deteg et al.  correspoiporters .L = 4.6, ψT = −3.4 mV, +]L = 167 mM, [Na+]L = 12 mM, and [Cl−]L = 166 mM, −/H+ exchanger (ClC-7unc and ClC-7ko) leads to a less acidic pH. In the ClC-7ko scenario, which mimics ClC-7 deficiency, we obtained the least acidification (pHL = 5) and the highest total membrane potential (ΔψT = 42.6 mV). The ClC-7unc led to a steady-state pHL of 4.9 and a positive total membrane potential (ΔψT) of 27.8 mV and sodium ([Na+]L), since they served as counter ions supporting acidification  because the only possible chloride transport pathway (ClC-7) was absent and remained low in our simulations of ClC-7unc ([Cl−]L = 29.6 mM), [Cl−]L increased to 166 mM for the ClC-7WT and ClC-7fast (WT (and ClC-7fast) lasted for 5000 s . Implementing all these possibilities would unavoidably increase the complexity of the model. This would restrict the detailed analysis of compensation mechanisms in perturbed scenarios. For simplicity, we opted to simulate the release channels solely via Ca2+ permeability.Differences in voltage dependent ClC-7 kinetics were hypothesized to be relevant during voltage jumps associated with lysosomal Ca release . To inve2+ is described asCa2+ is the passive flow through Ca2+ channel (positive for Ca2+ influx)The change in total CaCa2+ is the permeability per unit area for calcium ions, ([Ca2+f]i and [Ca2+f]e) are the modified luminal and cytosolic free Ca2+ concentration, respectively  to simulate the other ClC-7 scenarios  than the efflux observed for ClC-7WT and ClC-7fast (JCa2+ = −1.7 × 10−6fast and ClC-7WT (WT and ClC-7fast were about three orders of magnitude lower compared to ClC-7unc (JClC-7WT = JClC-7fast = −5.4 × 103 Cl−/s versus JClC-7unc = −950 × 103 Cl−/s)  for ClC-7WT and ClC-7fast scenarios. The opening of the Ca2+ channel did not result in changes of the ClC-7 driving force  activities.We observed an efficient Cacenarios b, accompcenarios d. Emptyi ClC-7WT . The max3 Cl−/s) i,j, caus only 4% f,g. We o2+ release pathway, we simulated the Ca2+ permeability with voltage- and pH-dependence as previously described for the prominent lysosomal Ca2+ release channel TRPML1 C = 0.6 mM) during 2 s, followed by Ca2+ release  during 2 s, as shown in Next, we investigated the impact of the different ClC-7 scenarios on subsequent cycles of lysosomal Ca2+ dynamics between WT and fast ClC-7, we simulated Ca2+ release with only ClC-7 as possible counter-ion conductance  under which conditions we had observed a small difference between the fast and wild-type ClC-7 scenarios  than those with ClC-7unc or ClC-7ko  show that even when the Ca2+ gradient is the same in the different scenarios, the Ca2+ release would depend on the counter-transport availability, which in turn differ between the different ClC-7 scenarios. Moreover, in 2+ via diverse potential mechanisms, which resulted in differential luminal Ca2+ concentrations in the different ClC-7 scenarios. Finally, the combination of Ca2+ uptake and subsequent release  and those without coupled transport (ClC-7unc or ClC-7ko). For the ClC-7unc and ClC-7ko scenarios, a reduced in lysosomal chloride concentration has been reported for ClC-7unc and ClC-7ko cell lines  has indeed been observed upon ClC-7 depletion or uncoupling c) showed that efficient Ca2+ uptake by this mechanism L to reach a steady-state level. For both simulated uptake mechanisms, lysosomes with a Cl−/H+ exchanger (in its wild-type or fast form), accumulated much more Ca2+ than those lysosomes with ClC-7unc and ClC-7ko. The reduction in the steady-state values for [Ca2+]L in lysosomes lacking Cl−/H+ exchange activity (ClC-7unc and ClC-7ko) is less pronounced when Ca2+ is taken up by a CAX with a 2:1 or 3:1 H+/Ca2+ stoichiometry, as compared to a CAX with a 1:1 stoichiometry, or when uptake is mediated by a channel from high extra-lysosomal Ca2+. Given the lower [Ca2+]L reached in ClC-7unc and ClC-7ko, our observation of a faster Ca2+ release without the Cl−/H+ exchange activity from lysosomes with the same Ca2+ concentrations (see above) may not be physiologically relevant. In the consecutive cycles of Ca2+ release and uptake, which we simulated in consistence with the hypothesis that lysosomal Ca2+ refilling may be triggered directly by lysosomal Ca2+ release [2+ concentration also leads to a decrease in Ca2+ efflux. This is in agreement with the experimentally observed reduced Ca2+ release from lysosomes with lowered Cl− concentrations [The Ca release , the redtrations .− from H+ transport, or reduced ion transport capability [2+-mediated process, yet the authors did not include Ca2+ in the model, nor explicitly considered the lysosomal compartment. Instead, the authors activate or de-activate V-ATPases and ClC-7 antiporters mimicking the fusion of the lysosomes with the ruffled border, which then results in the acidification of the pit and do not consider different osteopetrosis-associated ClC-7 mutations, unlike in our study.Dysfunction of ClC-7 leads to osteopetrosis and lysosomal pathology ,48. Thispability ,53,54,73pability . An exispability , refers 2+ uptake, both by CAX and by a Ca2+ channel, was unaffected by changes in the time-dependence of voltage-gated ClC-7 activation. Importantly, acceleration of the (de)activation kinetics did not affect Ca2+ release (unless we simulated an unlikely situation without Na+ and K+ channels) when it was simulated as a single event. When we simulated release and uptake cycles, consistent with the hypothesis that lysosomal Ca2+ refilling can be directly triggered by lysosomal Ca2+ release [2+ concentrations and peak Ca2+ release, which was stronger in the wild-type scenario  or expressing an uncoupled ClC-7 mutation (ClC-7unc) present underdeveloped ruffled borders [2+ release. Yet, the small differences generated by the acceleration of our in silico ClC-7fast, which mimics an extreme case of acceleration, compared to that observed for osteopetrosis-causing ClC-7 mutants [2+ release (due to H+ influx serving as part of the counterion transport) whereas triggered release of Ca2+ by NAADP was found to be paralleled by a lysosomal alkalinisation [To investigate how changes in ClC-7 (de)activation kinetics impinge on lysosomal ion homeostasis and dynamics, we implemented the time dependence of voltage-dependent ClC-7 (de)activation in our model. With this approach, we could in silico recapitulate electrophysiological measurements of the wild-type ClC-7 ,62,67,74 release , we genescenario . Lysosom borders ,53, like mutants ,59,60, a mutants . In fact mutants . This conisation . In addinisation ,14,78. T2+ and in the overall lysosomal ion homeostasis. It would be interesting, in future work, to couple this model with a mathematical model of the circadian clock [Our model enables a detailed analysis of the potential role of ClC-7 in the dynamics of Caan clock  and intran clock ,81,82 anan clock ,84,85,862+ uptake/release and to obtain a more mechanistic picture of lysosomal ion homeostasis.Moreover, a detailed experimental analysis will be possible with the development of novel ion and voltage sensors ,44,78,87