Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5  and Man4 . The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms. N-glycans, responsible for most of the N-glycan diversity, in order to produce only high-mannose N-glycans (Man4–Man9) is a powerful strategy to homogenize the N-glycan repertoire. N-acetylglucosaminyltransferase I (GnTI) is a key Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans (N-acetylglucosamine (GlcNAc) residue from UDP-GlcNAc to the acceptor substrate Man5 to produce GnMan5, which is a prerequisite for the subsequent action of all other processing enzymes  mutant lacks GnTI activity and cDNA sequencing identified the point mutation Asp144Asn, which introduced an extra N-glycosylation site impairing the GnTI folding   plants , predominantly Man5. Nonetheless, significant amounts of MMXF (9–3.8%), Man5F (3.1–0.4%), GnMXF (2.7–0%) or MMX(0–1.3%) on specific N-sites were also produced, suggesting the presence of a residual GnTI activity in these seeds  also lacked GnTI activity (cgl1-2 seeds showed only high-mannose glycans (The first ed seeds . The com folding . Early rn5 (75%) . However) plants . A decrereported . Many enysosomes . Hence, se seeds . The cglactivity . Analysi glycans , suggestgnt1 mutant line (SAI3G12) contains a T-DNA insertion in the sixth exon/intron junction of GnTI leading to the absence of native GnTI transcripts  (The rice nscripts . As a reosidase) , 2019.N-glycosylation pathway were selected from the Lotus japonicus retrotransposon 1 insertion population  in one line but sufficient for the synthesis of complex N-glycans in amounts comparable to WT plants as no impact on the N-glycan repertoire was observed. Complex N-glycans still accounted for 80% of total N-glycans  were obtained in the second study (N-glycans (73–85%), predominantly Man5, on the four N-sites, but also the presence of significant amounts of paucimannose N-glycans with β1,2-xylose and α1,3-fucose residues, suggesting a residual GnTI activity  suspension cells are characterized by a short culture cycle, easy scaling up in bioreactors, low risk of contamination by human pathogens and the possibility to purify secreted recombinant proteins directly from the culture medium  genes were inactivated simultaneously. This combined inactivation resulted in a BY-2 cell line with high-mannose N-glycans and no detectable α1,3-fucose. Generation of this BY-2 cell line capable of producing glycoproteins with a high-mannose profile is a powerful tool to improve our understanding of the impact of those N-glycans on the properties of vaccinal and biotherapeutic glycoproteins.Preventing e medium . BY-2 cee medium , a virale medium  or humane medium . In thisN. tabacum cv. BY-2 suspension cells ]. Cultures were grown in 50 mL of medium in a 250 mL Erlenmeyer flask and an 8% inoculum was transferred each week into fresh medium. Transformed cells were grown on MS agar plates supplemented with 100 μg/mL kanamycin. Samples used for genomic analyses and Western blot screening were harvested from liquid cultures obtained after at least two passages on solid selective medium (1–2 months) and at least two passages in liquid selective medium. Lines used for MS analyses were checked by Western blotting and the same profile was invariably observed.on cells  were groNicotiana tomentosiformis, N. sylvestris, and N. tabacum seeds were purchased from Bergerac Seed and Breeding. They were germinated and grown in soil under controlled conditions .Genomic DNA was extracted from 7-day-old BY-2 cells filtered on four layers of Miracloth  or from plant leaf material using the Wizard Genomic DNA Purification Kit (Promega).Genomic DNA was amplified by PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and the primers listed in RNA extraction was carried out using the Spectrum Plant Total RNA Kit (Sigma). cDNAs were obtained using M-MLV Reverse Transcriptase (Promega). PCR amplification was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs) and the primers listed in NtGnTI.A (AJ249882), NtGnTI.B (AJ249883), NsGnTI (XM_009803697 and XM_009776507), NtoGnTI (XM_009610507), FucTA (XM_016657530), FucTB (XM_016620229), FucTC (NM_001324945), and FucTD (XM_016585847).GenBank mRNA accessions are: GnTI were obtained by fusing the CRISPR RNA (crRNA) sequence (trans-activating crRNA (tracrRNA) optimized by NtGnTI orthologs. crRNAs were chosen to minimize the risk of off-targets using the Cas-OFFinder tool  targeting sequence  to the tder tool . Moreovepromoter .GnTI (GnTI.PTG) flanked with StuI and XhoI restriction sites, was synthesized (Genscript) and cloned into a pUC57 vector. The FucT/XylT.PTG of pPAM-FX-KO  genetic insulator. Complete sequence between left and right borders is given in The polycistronic tRNA-gRNA targeting AM-FX-KO  was replpromoter  and the r  was precipitated on 600 μg of 0.6 μm diameter gold beads (Bio-Rad) and the cells on the filter were bombarded under a 28-inch Hg vacuum (95 kPa), using a 1,100 PSI  rupture disk, at a shooting distance of 4 cm with a Biolistic PDS1000/He device (Bio-Rad). The filter was kept on solid MS medium for three days at 25°C under dark conditions. Filters were then transferred onto kanamycin-supplemented MS medium. After four to five weeks, the growing calli were transferred on new solid kanamycin-supplemented MS medium.Biolistic particle delivery was carried out with the plasmid pPAM-Agrobacterium tumefaciens LBA4404VirG-mediated transformation of BY-2 cells was carried out with the plasmid pPZP-GnTI/FucT-KO as described in 2EDTA, pH 8.0) supplemented with 1 mM phenylmethylsulfonylfluoride (PMSF), and protease inhibitor cocktail . Cell grinding was performed for 3 × 40 s at 5,000 rpm  with 2 min pauses on ice. The samples were centrifuged first for 5 min at 5,000 rpm at 4°C (Eppendorf Centrifuge 5417C), then for 7 min at 9,400 rpm at 4°C, and finally for 15 min at 54,000 rpm at 4°C . The protein concentration in the supernatant was quantified by Bradford assay and 12 μg TSCP were analyzed by reductive SDS-PAGE (4–20% polyacrylamide). Gels were either stained with Coomassie Brilliant Blue G-250  or transferred onto a PVDF membrane  for Western blotting. The PVDF membrane was incubated with the primary antibodies against β1,2-xylose  or α1,3-fucose  or a mix of both. Secondary horseradish peroxidase (HRP)-conjugated antibodies against rabbit IgG  were used for extracellular proteins samples. The signals were analyzed using a Kodak Image Station 4000R . HRP-conjugated antibodies were not used for TSCP analysis because of a strong signal attributed to intracellular endogenous peroxidase activity. Rather, secondary alkaline phosphatase-conjugated antibodies against rabbit IgG  were used.For extracellular protein glycosylation analysis by Western blot, 2 mL of a 7-day-old BY-2 culture in MS medium were filtered on three layers of Miracloth  and 40 μL of the filtrate were directly analyzed by reducing SDS-PAGE. Filtered BY-2 cells were used to harvest total soluble cellular proteins (TSCPs). Cell packs were transferred into a 2 mL micro tube (Sarstedt) containing 0.5 g of glass beads (0.85–1.23 mm) and 700 μL of homogenization buffer 2SO4 was slowly added to the culture medium with stirring at room temperature to 55% saturation and the solution was then kept for 2 h at 4°C and centrifuged at 3,000 g for 40 min at 4°C. The pellet was solubilized in 140 μL of PBS buffer and desalted on a PD-10 filtration column  equilibrated with PBS buffer.For the mass spectrometry analysis, cultures were grown in liquid D11b medium  without g for 5 min at 4°C, the supernatant was filtered on Amicon 3 kDa and the protein content assessed using the RC DC protein essay kit (Bio-Rad). Reduction of 350 μg proteins was performed in 50 mM (NH4)2CO3 and 10 mM of dithiothreitol at 56°C for 40 min at 650 rpm in a thermoshaker. Then alkylation of cysteinyl residues was performed using 20 mM C2H4INO at RT for 40 min at 650 rpm using a thermoshaker. 2D clean up purification kit  was used while the extra washing step (using wash additive) was skipped. Pellets containing proteins were resolubilized in 50 mM (NH4)2CO3 (pH 7.5) before trypsin digestion (protein:trypsin ratio of 50:1) at 37°C for 16 h at 600 rpm. The samples were freeze-dried using SpeedVac centrifugal evaporator and a second trypsin digestion was performed at 37°C in 80% acetonitrile using 100:1 protein:trypsin ratio for 3 h at 600 rpm. Trypsin was inactivated at 90°C for 2 min.Ribonuclease B from bovine pancreas was used as a positive control sample and was prepared using the same procedure as for the glycoprotein samples from BY-2 cells. The samples were first centrifuged at 8,000 N-glycans of interest, a two-step enzymatic release of the N-glycans was performed on the trypsin digested samples using first 3 U per 100 μg proteins of PNGase F  dissolved in 50 mM (NH4)2CO3 (pH 8) at 37°C for 16 h. Samples were freeze-dried and resolubilized in 48.3 mM C6H8O7 and 103.3 mM NaH2PO4.H2O buffer (pH 5). The second deglycosylation step was performed with 0.2 mU of PNGase A  per 100 μg proteins at 37°C for 16 h at 650 rpm. Samples were stored at −20°C after freeze-drying using a SpeedVac centrifugal evaporator.In order to guarantee the removing of all N-glycans fraction was separated from the peptide fraction using Waters Sep-Pack SPE C18 cartridges after being reconditioned using 5 mL of pure methanol, then 5 mL of 5% acetic acid, 5 mL of pure isopropanol and finally 15 mL of 5% acetic acid. Samples were loaded on the cartridge after solubilizing the dried residue in 200 μL of 5% acetic acid. N-glycans were eluted in the 3 mL of 5% acetic acid mobile phase. All the samples were freeze-dried and kept at −20°C. The N-glycans containing fraction was desalted using H-cartridges  after being reconditioned following the manufacturer recommendations. Desalted N-glycans elution was performed as follows: 3 mL of ultrapure water (discarded), 3 mL of 5% acetonitrile and 0.1% trifluoroacetic acid (discarded), and 4 × 0.5 mL of 50% acetonitrile and 0.1% trifluoroacetic acid. These collected fractions were freeze-dried and kept at −20°C.The N-glycans was then performed using a mixture of 31.5 mg of 2-aminobenzamide (2-AB) and 31.5 mg of NaBH3CN in 650 μL of 10 volumes DMSO: 3 volumes glacial acetic acid. Ten microliters of this mixture was added to the dried N-glycan fraction and incubated for 2 h at 65°C under 1,200 rpm agitation using a thermoshaker. The 2-AB labeled N-glycans were purified using S-cartridges  following the manufacturer recommendations and eluted using 3 × 0.5 mL of ultrapure water in Pyrex tubes. The samples were freeze-dried using centrifugal evaporator and kept at −20°C.The labeling of N-glycans were resolubilized in 10 μL of 50% acetonitrile spiked with 0.1% formic acid and vigorous vortexed in 0.6 mL Eppendorf tube. One microliter of 2-AB labeled N-glycan and 1 μL of 2,5-dihydroxybenzoic acid solution  were mixed and spotted (dried-droplet method) on a MALDI plate Anchorchip384BC fitted on its plate adapter (Bruker) and dried at RT. MALDI mass spectra were obtained using a SolariX XR 9.4T FT-ICR (Bruker) fitted with the dual ESI/MALDI source and Smartbeam LASER. The mass range was set from 150 to 3,500 m/z using 4 megawords acquisition, resulting in an average mass resolving power (FWMH) of about 150,000 at m/z ≈1,100. Two hundred laser shots per scan in small focus mode were selected at the rate of 2,000 Hz. Red P . Only peaks with an intensity higher than 1.4 × 106 were considered. The ion mass lists were manually generated based on the GlycoMod tool (N-glycans1. Mass accuracy (0.1 Da) was used to refine the list of identified N-glycans and to manually annotate the mass spectra.The purified 2-AB labeled z. Red P  was usedMod tool , 2003 usN. tabacum is an allotetraploid species resulting from the cross between N. tomentosiformis and N. sylvestris  as well as the N. tomentosiformis and N. sylvestris genomes2 identified the GnTI.A full-length gene in all four N. tabacum cultivars (Ntab-TN90_AYMY-SS16267) and in N. tomentosiformis (Ntom_KB953441.1). On the other hand, the GnTI.B gene was found to be split into three contigs in N. tabacum TN90 and BX cultivars , and in two contigs in N. sylvestris (Nsyl_KD955459.1: exons 1-11 and Nsyl_KD976298.1: exons 12-19). Both NtGnTI genes were also annotated in NCBI database: NtGnTI.A (NCBI gene ID: 107789589) consists of 19 exons spanning on a 12.5 kb genomic sequence (NtGnTI.B (gene ID: 107800221) is incomplete since the annotated contig covers only the exons 1 to 10.lvestris . The NtGT1 genes , and NtFlT genes . The NtGibraries . Blast asequence , while NGnTI.A and GnTI.B genomic regions between exons 11 and 12, genomic DNA was extracted from the three Nicotiana plants as well as from BY-2 cells. Next, the GnTI region spanning exons 6 to 15 was amplified using non-specific primers [Ex6F and Ex15R]  were identified, showing the presence of numerous sequences highly similar to NtGnTI.B intron 11 in the N. tabacum genome. In silico analysis of NtGnTI.B intron 11 sequence revealed the presence of three ORFs of 447, 183, and 507 codons  whereas NtoGnTI displayed a single mutation (Thr175Ala), compared to the database sequence (XM_009610507).To assess the expression of both ortholog . GnTI.A-seq data  in whichterature . In addiNtGnTI.A and NtGnTI.B by targeting exons 1, 3, 13, and 14 (GnTI.PTG). Once the PTG is transcribed, individual gRNAs are expected to be released by the endogenous tRNA processing machinery  were further analyzed using either anti-β1,2-xylose or anti-α1,3-fucose antibodies  were designed to inactivate both , and 14 . A transachinery . The GnTzed Cas9  to gener plasmid . BY-2 ce control . Four Gntibodies . While nblotting .GnTI.PTG genetic construct was combined with the FucT.PTG construct previously used to obtain the XylT/FucT-KO cell line  for which no signal had been observed were chosen for further analysis. Secreted glycoproteins were analyzed by Western blotting using either anti-β1,2-xylose or anti-α1,3-fucose antibodies , GnGnXF (20.9%), and GnMX (14.6%). Complex, paucimannosidic and hybrid N-glycans accounted for 84.8% of the N-glycans on proteins secreted in WT BY-2 cells. High-mannose N-glycans made up for only 15.2% of the N-glycans. GnTI inactivation resulted in a dramatic shift toward high-mannose N-glycan structures in GnTI-KO#2 and GnTI/FucT-KO#10 lines, in which they accounted for 98.4% and 99% of the N-glycans, respectively. Only six N-glycan structures, corresponding to Man3-8 were identified in the KO lines. The main N-glycan structures detected in GnTI-KO#2 and GnTI/FT-KO#10 were Man5  and Man4 .Secreted glycoproteins of WT,  control . FourteeGnTI alleles, genomic DNA was extracted from WT, GnTI-KO#2, and GnTI/FucT-KO#10 cell lines. The GnTI region encompassing the four targeted loci (exon 1 to exon 14) was PCR-amplified using primers hybridizing to conserved regions [Ex1F and Ex15R]  for GnTI/FucT-KO#10  of α1,3-fucose residues as compared to WT secreted proteins. Absence of N-glycans containing β1,2-xylose residues demonstrates the suppression of the entire GnTI activity since GnTI-processed N-glycans would be recognized as substrates by Golgi-resident glycosyltransferases like xylosyltransferases  was carried out to generate GnTI/FucT-KO cell lines. Secreted and soluble intracellular proteins of three GnTI/FucT-KO cell lines  were totally devoid of any β1,2-xylose and α1,3-fucose residues.Complete inactivation of sferases . Genomicresponse . To remoN-glycan structures of secreted glycoproteins from WT, GnTI-KO#2 and GnTI-FucT-KO#10 lines identified the major changes that occurred in the KO lines. While most N-glycans in the WT cell line were of complex type, none were detected in either of the KO lines. Man4 and Man5 structures accounted for 96.1 and 96.9% of the N-glycans in the GnTI-KO#2 and GnTI-FucT-KO#10 cell lines, respectively. Unlike the Western blotting data, we could not detect any fucosylated structure in the GnTI-KO#2 cell line. However, this can be explained by the fact that the α1,3-fucose signal observed by Western blotting in GnTI-KO#2 cell line was very weak. It probably belongs to fucosylated N-glycans distributed between different masses. Their individual MS signal drops below the detection limit of the method.Mass spectrometry analysis of N-glycans in GnTI-KO cell lines was surprising and can only be explained by a relaxed substrate specificity of FucTs, unlike what is usually reported in the literature. Our hypothesis is that at least one of the four N. tabacum FucTs can recognize and process high-mannose N-glycans, although with a lower rate than hybrid and complex N-glycans. The lack of high-mannose N-glycans with α1,3-fucose reported in most plant and plant cell studies is probably due to their very low levels. It could also be correlated with a higher affinity of FucTs for complex N-glycans. However, detection of Man5F N-glycans on human glucocerebrosidase and α-iduronidase produced in the A. thaliana seeds of the conditional cgl1-1 mutant has been reported several times . Core α1,6-fucosylation by FUT8 in mammals was first believed to be strictly GnTI-dependent because α1,6-fucosylated N-glycans identified by mass spectrometry were for the overwhelming majority hybrid or complex types. Furthermore, in vitro studies showed that FUT8 requires the presence of an α1,3-arm GlcNAc  to use free N-glycans as substrates . This N-glycoprofile is strongly recommended for some therapeutic glycoproteins (N-glycan homogeneity can ease the protein purification as well as the process reproducibility, leading to more consistent therapeutic efficacy (N-glycan repertoire, such as the humanized XylT/FucT-KO cell lines (proteins . Furtherefficacy . As a rell lines  or the tftp://massive.ucsd.edu/MSV000086640/.The original contributions presented in the study are publicly available. This data can be found here: Repository MassIVE: MSV000086640, XH and CN: conceptualization. XH and JF: methodology. XH, JF, AC, LB, and CN: investigation. XH, JF, and CN: data curation. XH: writing – original draft preparation. CN, JF, and FC: writing – review and editing. CN, LQ, and FC: supervision. FC and EDP: funding acquisition. All authors have read and agreed to 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.


Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the  2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.Many viruses use specific viral proteins to bind calcium ions  or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly. Orbivirus genus in the Reoviridae family is an insect-borne animal pathogen. BTV is vectored by Culicoides spp. and causes infection in vertebrate hosts  in many parts of the world that has considerable economic impact. The nonenveloped BTV particle is a complex icosahedral structure, consisting of seven structural proteins (VP1 to VP7) that are organized in an outer capsid and an inner capsid (core). The outer capsid is composed of two major proteins, VP2 and VP5, and is responsible for attachment and membrane penetration. Both proteins are lost during endocytosis, and the inner core is subsequently released into the cytoplasm. The BTV core consists of the remaining five proteins and the viral genome of 10 double-stranded RNA (dsRNA) segments. In addition to 7 structural proteins, 4 nonstructural proteins, NS1 to NS4, are synthesized during virus replication. Two of these are major nonstructural (NS) proteins, NS1 and NS2, which are synthesized during early infection, and each plays an essential role in virus replication. The third NS protein, NS3/NS3A, is a transmembrane protein and facilitates release of the newly assembled BTV  of the oteins VP to VP7 tbled BTV 3. NS4 isled BTV 46. Previooteins VP to VP7 tfe cycle , 9. For 2+ binding motif. We identified EF-hand-like motif in NS2, which is the only viral encoded phosphoprotein and is essential for replication  analysis of NS2 oligomer in the presence of Ca2+ exhibited a helical architecture. By site-specific targeted mutagenesis in the recombinant NS2 and in the replicating viral genome by reverse genetics, we identified the specific Ca2+ binding site of NS2 and demonstrated its importance in NS2 phosphorylation level, formation of VIBs, and virus replication. Altogether, our results suggest that Ca2+ sensing by NS2 influences NS2 phosphorylation and may be involved in the regulation of VIB assembly/disassembly, a process critical for virus replication and the release of newly assembled cores from VIBs  and four NS proteins , thus making this putative motif less obvious. These acidic residues in NS2 are continuous rather than alternate as found in the case of standard EF-hand motifs (2+ bowl” found in BK (big potassium) channels (–2+ binding motif is likely to be important for BTV replication (In order to identify putative Caproteins . In our erfamily . In parterfamily  13–14).–14.2+ bid motifs . These cchannels –21. Furtlication .Escherichia coli in a nonphosphorylated form that was purified and analyzed by SDS-PAGE gel . The changes in intrinsic far-UV CD spectra were recorded, as the direct measure of Ca2+ binding to purified NS2, without (apo) or with (holo) Ca2+ at increasing concentrations of Ca2+ from 5 μM to 10 mM to calculate the dissociation constant, dK. NS2 showed a dK value of 53.9 (±8.4) μM for Ca2+ binding. In comparison, another divalent ion, Mg2+, when tested for NS2 binding in a parallel titration experiment with the same concentration range, showed a dK value of 2.48 (± 0.4) mM, suggesting a weaker binding of Mg2+ compared to Ca2+ , NS2 showed a very different CD spectrum . Im. Im2+ onspectrum  suggestilmodulin . The chaing site  and B , ED256–257AA (A2), and DDE260–262AAA (A3), respectively, were then purified and analyzed by gel electrophoresis to determine that each mutant protein expressed was stable and equivalent to wild-type NS2 (wtNS2)  similar to that of wtNS2 , suggesting not much change in the Ca2+ binding activities of mutants A2 and A3  . Further program . There w program . CD titr2 and A3  and D. H ∼150 μM  and D. T2+ binding site (aa 250 to 253) is in juxtaposition of serine residues , known for phosphorylation modification, it was more likely that Ca2+-mediated transition in the secondary structure elements could affect the level of NS2 phosphorylation. Therefore, we undertook an in vitro kinase assay for NS2 phosphorylation, a modification required for VIB assembly  in the presence of Ca2+ ions . To confirm further the specificity of Ca2+ binding activity on NS2 phosphorylation, we assessed the three NS2 mutants A1, A2, and A3 as substrates for CK2α kinase assay in the presence of Ca2+ . In total, 159,361 particle images were selected from 2,712 CryoEM micrographs and subjected to image classification to obtain two-dimensional (2D) class averages. Particles with clear helical organizational features were observed in some 2D classes  as described previously (250–253AAAA), A2 (ED256–257AA), or A3 (DDE260–262AAA). In addition, two multisite mutations in S8, A1 + A2 (DDDE250–253AAAA + ED256–257AA) and A1 + A2 + A3 (DDDE250–253AAAA + ED256–257AA + DDE260–262AAA) were created  were previously substituted by alanine residues (SAA) that perturbed virus replication (10), suggesting no apparent change due to these mutations , A2 (ED256–257AA), or A3 (DDE260–262AAA) recovered from the NS2 complementary BS8 cells, and VIB morphology in the infected BSR cells was visualized by confocal microscopy. Changes in the average size (area in μm2) of VIBs in cells infected with wt and mutant viruses were quantified. Cells infected with A1 mutant virus showed smaller VIBs compared to the VIBs in the wt virus-infected cells; however, mutants A2 and A3 showed no significant change in average size of VIBs  residues identified for Ca of VIBs . The A1  of VIBs . Taken tsay data  and 6.2+ binding motif resulting in diminished VIBs was due to poor phosphorylation of NS2, BSR cells were infected with wt and the mutant viruses in addition to NS2 phosphorylation-negative mutant SAA following optimized protocol for BTV , specifically interferes with NS2 phosphorylation in cells infected with Ca2+ mutant virus.To obtain direct evidence on whether the defective Ca for BTV . BSR cel for BTV . Pulldow for BTV  11). Th. Th2+ bi from CD  and 6. T2+ binding proteins containing EF-hand-like motifs  is highly specific. Our data also suggest that the unusual Ca2+ bowl-like motif of NS2 acts as a Ca2+ sensor. In addition, in the presence of a specific chelating agent, such as EGTA, the CD spectrum of NS2-Ca2+ was partially reversed. Further, like several other CaBP, NS2 also demonstrated a contrasting change in the percentage of helix in NS2 (helix-to-coil) upon Ca2+ binding in far-UV CD spectroscopic measurements. The Ca2+ concentrations used in the far-UV CD experiments were to a 1:1 stoichiometry (approximately) with NS2, which is a decamer in purified form. More importantly, when we introduced the same mutations into the NS2-encoding gene of a replicating viral genome, the A1 mutation failed to recover infectious virus and plaque formation, reflecting the importance of the Ca2+ sensing activity of NS2 for the production of infectious virus.Several viruses are known to synthesize Cae motifs . Our in in vitro (recombinant NS2 protein) and in vivo (NS2 expressed by mutant virus particles in cells). In our in vitro kinase assay, the level of phosphorylation was increased in the presence of Ca2+, indicating that Ca2+-mediated changes in secondary structural elements in NS2 might have led to better access to the phosphorylation sites to the enzyme. The data from the kinase assay was consistent with the CD data on Ca2+-mediated changes in secondary structural elements (helix) in NS2. These Ca2+-induced changes in NS2 are significant, although they do not appear to destabilize its oligomeric state as revealed by CryoEM analysis and size exclusion chromatography. This suggests that it is the oligomeric NS2 that senses the Ca2+ ions, possibly through coordination between the protomers, rather than individual monomeric subunits independently sensing Ca2+ ions and forming a decamer. Interestingly, a ring-like shape was inferred for full-length NS2 based on negative-stain electron microscopy  do the same to Ca2+ pumps at the endoplasmic reticulum  during the assembly of VIBs, the viral assembly factories.Several viral proteins are reported to alter cellular calcium homeostasis to assist viral life cycle by modulating mainly membrane-associated Camembrane , viral pembranes , and NSPeticulum . It is nReoviridae family also form similar VIB-like structures , although each have their own unique features , ED256-257AA (CGATGATGAACAAGGTGCGGCTGCGAGTGACGATGAAC), and DDE260-262AAA (CAAGGTGAGGATGCGAGTGCCGCTGCACACCCAAAAACTCATA). Obtained mutants were sequenced using internal NS2 primers in order to confirm the presence of the desired mutation(s). The wild-type NS2 and mutant proteins were expressed in the E. coli bacteria strain BL21(DE3) pLysS. The culture was grown at 37°C until optical density at 600 nm (OD600) reached 0.5 to 0.6 and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The bacterial cultures were grown at 37°C for the next 4 h postinduction. Cells were then lysed for the protein purification. Ni-nitrilotriacetic acid (Ni-NTA) affinity purification was used to purify the wild-type and mutant proteins eluting in the presence of 250 mM imidazole . In order to remove traces of nucleic acid from the purified protein, samples were treated with Benzonase nuclease or micrococcal nuclease where necessary. For gel filtration, a Superdex 200 10/300 GL (GE/Cytiva) column was used in a running buffer of 20 mM HEPES, 100 mM NaCl, pH 7.4, at a flow rate of 0.2 ml/min.Based on the SMART motif search results , the pla2+ salts in increasing concentration. Far-UV CD spectrum data were collected from 260 to 195 nm with a 0.5-mm rectangular cell path length at 20°C on the Applied Photophysics Chirascan and Chirascan Plus spectrometers  attached to a Peltier unit (Quantum Northwest TC 125). The UV and CD spectra were smoothed  and analyzed using Origin v6 and APL Pro-Data Viewer v4.2.15. For comparative metal ion titration experiments, same preparation of the wtNS2 protein sample was aliquoted in two halves just before titration on CD instrument. Equal amounts of Ca2+ and Mg2+ were added to each tube, respectively, in the increasing concentrations to obtain same concentrations for two metal ions. The Ca2+ or Mg2+ concentrations were taken from the 5 μM to 10 mM range. For dK measurement, CD222 nm was plotted (y axis) for each Ca2+ concentration (x axis), and the dK value was derived using GraphPad Prism choosing inbuilt one-site total function for nonlinear regression (curve fit). Likewise, similar concentrations of mutant proteins A1, A2, and A3 were used for titration, and a similar Ca2+ concentration range (5 μM to 10 mM) was studied.The circular dichroism (CD) spectra of wild-type apo NS2  were recorded in CD buffer  at room temperature (20°C). All buffers were pretreated with Chelex 100 resin to remove divalent metal ions contaminants. For calcium binding studies, the apo NS2 sample was titrated with CaE. coli cells and purified using nickel and glutathione Sepharose, respectively. The kinase reaction was conducted in a 50-μl reaction mixture in 1× reaction buffer . The reaction tubes were prepared by adding two different concentrations of Mg2+ or Ca2+ while keeping the substrate (0.7 mg/ml) and enzyme (0.4 mg/ml) concentrations unchanged. For other tubes, different mutants of NS2 were added with Ca2+. The reaction was started by adding 10 μCi of [γ-32P]ATP  and 0.5 mM ATP in each tube at an interval of 15 s. Additional 1× buffer was added to the tubes to adjust the dilution factor, making each tube contain up to 50 μl of reaction volume. The reaction tubes were then incubated for 30 min at 37°C. After incubation, the reaction tube was boiled in SDS sample buffer and loaded on SDS-PAGE to run at 90 V for 3 h. The SDS-PAGE gel was then dried, exposed to film, and observed under imager.For the kinase assay, substrate protein NS2 (6×His tag) and kinase enzyme CK2 (GST tag) were expressed separately in 2, was applied to thin continuous carbon film on lacey grid (Ted Pella) and blotted using a Vitrobot Mark IV (Thermo Fisher Scientific) with the standard Vitrobot filter paper at 4°C. The blotting time was set to 6 s, blotting force was set to 2, and drain time was set to 1 s. The grid was flash-frozen in liquid ethane and stored in liquid nitrogen. A total of 2,712 micrographs were collected on a Titan Krios 300 kV electron microscope (Thermo Fisher Scientific) equipped with a Gatan imaging filter (GIF) Quantum LS and a Gatan K2 Summit direct electron detector operated in super-resolution mode at magnification of 130K . The GIF slit width was set to 20 eV. The dose rate on the camera was set to ∼6.5 electrons/pixel/s. The total exposure time of each movie was 8 s, which fractionated into 40 frames of images with 0.2-s exposure time for each frame. Dose-fractionated frames were 2× binned  and aligned for beam-induced drift correction using UCSF MotionCor2  by reverse genetics as previously described  using mMESSAGE mMACHINE T7 Ultra kit (Ambion) as previously described  using EndoFectin (GeneCopoeia) overlaid with 1% agarose and incubated 3 days at 35°C in 5% CO2. Visible plaques were picked up and resuspended in 1% FBS containing medium, and/or cells were subsequently fixed with 10% formaldehyde and stained with crystal violet. Each recovered virus was plaque-purified, amplified, and harvested 3 days postinfection. Viruses were titrated using plaque assay.BSR cells (BHK-21 subclone) or BS8 (BSR cells stably expressing NS2/segment of BTV) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (vol/vol) fetal bovine serum (FBS) at 35°C in 5% COescribed . Each reescribed . Reverseescribed . Brieflyx, 1,912; y, 1,912; 12-bit). Each field contained in average of 17 infected cells, and the experiment was repeated three times independently. Maximum intensity projection of each z-stack was performed using Zen software, and obtained images were further processed using ImageJ software . Only particles  with a size >0.5 μm2 were selected for particle analysis. The experiment was performed three times, and in total 1,600 VIBs were used for the wild-type virus, 764 VIBs for the DDD250-252AAA mutant, 1,534 VIBs for the ED256-257AA mutant, and 1,597 VIBs for the DDE260-2AAA mutant virus, respectively.BSR cells were grown on coverslips and infected at a multiplicity of infection (MOI) of 1 with NS2 wild-type or mutant-recovered viruses. Twenty-four hours postinfection, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) solution, permeabilized with 0.5% Triton X-100 (Sigma), blocked with 1% bovine serum albumin (BSA) (Sigma), and subsequently stained using a guinea pig anti-NS2 primary antibody (lab made) and an anti-guinea pig Alexa 488 coupled secondary antibody (Thermo Fisher Scientific). Nuclei were stained using Hoechst 33342 (Thermo Fisher Scientific). Images were acquired using an ×100 oil objective and a Zeiss Axiovert LSM 880 confocal microscope supplied with the Zen software. For each infection condition  , five fi2, 25 mM NaF, 1 mM Na3VO4, 1 mM beta-glycerophosphate, and 10 mM sodium pyrophosphate and protease inhibitor) for 30 min on ice. Lysates were centrifuged at 800 g for 15 min. Supernatants were recovered and added to protein A Sepharose beads conjugated to guinea-pig anti-NS2 and were incubated on ice overnight. Samples were centrifuged at 2,000 × g for 2 min. The supernatant was removed and the protein A Sepharose beads washed with lysis buffer. Samples were centrifuged at 2,000 × g for 2 min. This wash process was repeated 4 times. SDS loading buffer was then added to the protein A Sepharose beads before being boiled. SDS-PAGE gels were stained with Pro-Q Diamond phosphoprotein gel stain (Thermo Fisher). For stain, the respective fluorescence was detected and quantified.NS2 was purified from BTV1-infected BSR cells (MOI = 1) after 18 h. Cells were washed with phosphate-buffered saline (PBS) before lysis. Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 125 mM NaCl, 5% Glycerol, 0.2% NP-40, 1.5 mM MgCl


Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca Several tissues express CaCCs that participate in vital physiological functions5. Thus, a role for TMEM16A and TMEM16B in smooth muscle contraction, control of blood pressure, control of gastrointestinal movements, regulation of cardiac and neuronal excitability, fluid secretion in exocrine glands, secretion of melatonin, mucin and insulin, sperm capacitation and motility, inhibition of polyspermy, and sensory transduction was established using tissue-specific knockout mice16. In addition, TMEM16A modulates the partitioning of membrane phosphoinositides and endocytic transport by controlling the [Cl−]i17. Overexpression of TMEM16A is associated with hypertension, increased cell proliferation and cancer progression21.Anoctamin-1 (ANO1 or TMEM16A) and Anoctamin-2 (ANO2 or TMEM16B) are the pore-forming subunits of Ca2+ ions to the channel when the intracellular Ca2+ concentration ([Ca2+]i) increases26. Structural and mutagenesis analysis show that Ca2+ ions bind to an acidic Ca2+ pocket formed by four Glu, one Asp and one Asn27. The pocket is located near the cytosolic side facing the permeation pathway. However, other divalent cations and maybe trivalent cations too can support TMEM16A activation. Based on the cation concentrations to obtain the half-maximum response, the cation selectivity of TMEM16A gating machinery is Ca2+»Sr2+»Ba2+»Cd2+,29. Gd3+ may also activate TMEM16A since its application removed the inward rectification of the Gly644Pro TMEM16A mutant channel30. Mg2+, the most abundant divalent cation in the cytoplasm of our cells31 is unable to activate TMEM16A, however, Mg2+ competes with Ca2+ and decreases the apparent Ca2+ sensitivity of TMEM16A29. Despite its low cation selectivity, the Ca2+-binding pocket of TMEM16A does not interact with monovalent cations. Due to its acidic chemical nature, the Ca2+-binding pocket is prompt to protonation. In fact, intracellular H+ compete with Ca2+ for these acidic residues. By doing so, H+ decreases the Ca2+ affinity of TMEM16A causing a reduction in channel activity33. Thus, intracellular H+ interact with the pocket, but can this interaction prompt TMEM16A activation? In this work, we demonstrate that voltage activation of TMEM16A in the absence of intracellular Ca2+ is due to voltage-dependent titration of the Ca2+-binding pocket. In the absence of Ca2+ voltage induced strong outwardly rectifying anion currents sensitive to TMEM16A inhibitors. These currents activate and deactivate very fast (<1 ms) and displayed little time dependence. Furthermore, we show that Ca2+-independent activation of TMEM16A under acidic conditions resulted from voltage-dependent protonation of Glu/Asp residues.Activation of CaCCs is triggered by voltage-dependent binding of two Ca2+, we recorded whole cell currents from HEK-293 cells expressing TMEM16A dialyzed with a solution containing 25.24 mM EGTA without Ca2+. Under this condition, a current induced by voltage (Vm) is likely to result from gating of TMEM16A and we will refer to it as Vm-activated TMEM16A current or ICl,Vm. Figure Cl,Vm recorded from a cell stimulated with the Vm protocol shown in Fig. Cl,Vm activates and deactivates very fast. The time constant of activation was 0.59 ± 0.11 ms at +160 mV (n = 5); no tail currents were recorded upon repolarizing at −100 mV. The magnitude of ICl,Vm was constant during the entire stimulus duration. This behaviour contrasted with the classical time-dependent ICl generated upon depolarization and the corresponding large tail currents induced by repolarizing to −100 mV in the presence of 0.2 µM Ca2+ . The magnitude of ICl,Vm was small; at +160 mV, it was about 10 times smaller than that observed in the presence of 0.2 µM Ca2+ (blue vs black). In contrast, TMEM16B, a paralog of TMEM16A, was activated in cells dialysed with 2.5 µM Ca2+ but not in cells dialysed with 25.24 mM EGTA and 0 Ca2+  obtained from cells dialysed with 0 Ca2+ and exposed to the indicated anions on the extracellular side. Because ICl,Vm displays strong outward rectification, we could not reliably measure reversal potentials to determine the anion selectivity sequence from permeability ratios. However, by taking the ratio of IX,Vm (the current carried by anion X) relative to ICl,Vm at +160 mV we obtained the following anion selectivity sequence: SCN− , I− , NO3− , Br−, and Cl− . This sequence was the same as that obtained in the presence of 0.2 µM Ca2+  acids, blockers of TMEM16A. Figure 2+ solutions and exposed to increasing concentrations of tannic and A-9-C acids. ICl,Vm was inhibited with IC50 values of 18.2 ± 1.7 µM and 0.98 ± 0.22 mM, respectively. The IC50 values determined for both inhibitors are like those reported in the presence of Ca2+ 35. Finally, we recorded from mock-transfected HEK-293 cells dialyzed with 0 or 0.2 µM Ca2+ but we did not observe endogenous currents activated by Vm , HEPES (7.3), MES , citric acid (5.0), or tartaric acid (4.0). No difference in channel activation was observed between data obtained with different buffers at the same pH. Figure Cl,Vm recorded at −100, −40, +20, +60, +120 and +160 mV using the protocol shown in Fig. Cl,Vm recorded at the indicated pH (left column), traces are representative of 5 independent experiments. As pHi decreased from 8.0 (top) to 4.0 (bottom), the magnitude of ICl,Vm increased. Under acidic conditions ICl,Vm activated and deactivated very rapidly. At +160 mV, the time constant of activation was 0.24 ± 0.02 ms at pHi = 4 (n = 5). Tail currents were nearly absent but at pHi 4.0 a small and fast inward current became evident (inset shows a magnification of the tail current). The corresponding ICl,Vm - Vm relationships are shown on the right column of Fig. i from 6 to 4.0 produced a 5-fold increase in ICl,Vm  vs Vm at different [H+]i for two reasons. First, the reversal potentials (Vr) measurements at pHi ≥ 6.0 were unreliable due to ICl,Vm rectification . Second, the instantaneous current-voltage relationship after a depolarization to +160 mV was not linear  were the same for steps between −100 to +160 mV as if the open probability was Vm-independent.Under physiological i increase the magnitude of ICl,Vm is increased. Unfortunately, only at +160 mV and pHi 4.0, we obtained a hint of saturation. Experiments at pHi 3.0 were not possible because the cells died quickly. Thus, to determine the equilibrium constant of protonation (K) at each Vm, we first fitted the data collected at +160 mV to a Hill equation . Continuous lines in Fig. 2 values show that this fitting procedure is a good quantitative description of the experimental data at all Vm. Figure pKa (= −log10K) values as a function of Vm. At positive Vm, the pKa value increased indicating that less H+ are required to generate 50% of ICl,Vm. This Vm dependence of pKa resembles the Vm dependence of EC50 for intracellular Ca2+ 22. The relation pKa – Vm was fit with Eq. K0 . A pK0 value of 0.97 ± 0.07 and a δ value of 1.39 ± 0.04 were estimated this way. We conducted similar experiments with inside-out patches; unfortunately, most patches were unstable at acidic conditions. Figure i 8.0, 6.0, 5.0, and 4.0. The pKa obtained from inside-out patches has a similar Vm-dependence as the pKa obtained from whole cell recordings. In this case, the average pK0 and δ values were 1.77 and 0.79, respectively. Thus, taken together our results support the hypothesis that Vm gates TMEM16A due to intracellular protonation.To advance the hypothesis that Vm activation of TMEM16A is due to protonation we must show that the equilibrium constant of protonation  then Vm would activate Gln mutants at pHi 7.3 without the need for acidification. Figure Cl,Vm recordings at −100, −40, +20, +60, +120 and +160 mV from five independent cells expressing (from top to bottom) WT, E702Q/E705Q (2 M), E702Q/E705Q/E734Q (3 M), E702Q/E705Q/E734Q/D738Q (4 M), and E654Q/E702Q/E705Q/E734Q/D738Q (5 M) channels. All cells were dialyzed with 25.24 mM EGTA/0 Ca2+ and pHi 7.3. As the number of mutations accumulates, the magnitude of ICl,Vm increased mirroring the effect of intracellular acidification. ICl,Vm onset was fast, had no time dependence and no tail currents at −100 mV. Very fast tail currents were observed only with the quintuple mutant (see magnification in the lower-left panel). The corresponding ICl,Vm - Vm relationships  in the Ca2+-binding pocket increased  induced a 6.5-fold increase in ICl,Vm in 5 M channels compared to WT.To identify Glu and Asp residues within the Cased Fig. . At pHi 2+-binding pocket into Gln allowed activation of TMEM16A at pHi 7.3 in the absence of intracellular Ca2+. If these residues are the main source of Vm sensitivity under acidic conditions, then increasing the [H+]i should have little or no additional effect on ICl,Vm, and the Vm dependence of pKa should be abolished. To test these predictions, we recorded ICl,Vm from cells dialyzed with an intracellular solution with pHi 4. Figure Cl,Vm recordings for WT, 2 M, 3 M, 4 M, and 5 M channels (left to right). Current kinetics of ICl,Vm at pHi 4.0 and 7.3  at pHi 4.0. The corresponding ICl,Vm - Vm curves displayed less rectification  or inside-out patches (right). Unlike WT channels , indicating that residues other than Glu and Asp are being protonated. These pKa – Vm curves were fitted with Eq. pK0 and Vm sensitivity. pK0/δ values were 4.69 ± 0.08/0.13 ± 0.04 (inside out) and 3.91 ± 0.14/0.70 ± 0.07 (whole cell). Thus, compared to WT (replotted in orange) protonation in 5 M channels is less Vm-dependent. This loss of Vm dependence is also illustrated by the fact that this channel is partially open at a low [H+]i. Thus, mutating the acidic residues decreased the Vm dependence of TMEM16A titration lending support to the idea that the Ca2+-binding pocket is the target of intracellular H+. In addition, the activity of TMEM16A-5M channels was enhanced under acidic conditions by a weakly Vm-dependent protonation mechanism.To investigate if the Vm dependence of els Fig. , the 5 M+ activates TMEM16A, we investigated whether protonation of the Ca2+-binding pocket increases the open probability. To this end, we recorded ICl,Vm - Vm curves from inside-out patches consecutively exposed to pHi 7.3 and 4.0. We used a ramp protocol that changed the Vm between −100 to +200 mV in 635 ms. pHi 4.0 was chosen because under this acidic condition 64% of the time the Glu side chain will be in the protonated state, assuming the pKa value of Glu is 4.25. Figure Cl,Vm - Vm curves recorded from patches excised from a cell expressing WT (left) or 5 M (right) channels. At pHi 7.3, the WT channels displayed a small ICl,Vm at all Vm range. However, upon exposure to a solution pHi 4.0 the ICl,Vm was strongly enhanced resulting in an outward rectifying current. Similarly, in 5 M channels (right) ICl,Vm increased further when the pHi went from 7.3 to 4.0 with little alteration in the reversal potentials (ΔVr= 4.95 ± 0.8 for WT and ΔVr = 4.9 ± 0.9 mV for 5 M). To determine whether the open probability was enhanced by acidification, we calculated the ratio of ICl,Vm at pHi 4.0 and 7.3 (IpHi = 4/IpHi = 7.3). This value is directly proportional to the changes in the open probability induced by the acidification at each Vm. These ratios are shown in Fig. pHi = 4/IpHi = 7.3 for 5 M channels was Vm-independent (orange), albeit acidification produced a 3.5-fold increase in the open probability in the −100 to +200 mV range. Therefore, protonation is enough to grant Vm-dependent activation to TMEM16A channels in the absence of intracellular Ca2+. Also, the titration data indicates that TMEM16A activity is enhanced by Vm-independent protonation.To further understand the mechanism by which intracellular H2+ is enhanced by mutating the acidic residues of the Ca2+-binding pocket. We took advantage of the strong activation of 4 M and 5 M channels at pHi 7.3 to record tail currents to analyse the effect of acidification on voltage-dependent activation. We recorded tail currents at +100 mV using a P/8 protocol in cells expressing WT, 4 M and 5 M channels dialyzed with an internal solution pHi = 4.0. Because ICl,Vm reaches steady-state very fast, we employed 20 ms depolarization pulses . V0.5 values were the same for WT, 5 M and 4 M channels  we can conclude that intracellular protons shifted the Vm activation by more than −100 mV.The data described show that the activation of TMEM16A in the absence of intracellular Caels Fig. ; these v2+ can be prompted by strong depolarizations, an elevation in temperature, mutating residues Ile637 and Gln645 in the sixth transmembrane segment (Ile641 and Gln649 in our clone) and deleting EAVK segment in the first intracellular loop38. To explain these results, an intrinsic Vm sensitivity in TMEM16A channels has been proposed37. However, in TMEM16A an obvious Vm-sensing domain is lacking. Since intracellular acidification reduces TMEM16A activity due to competition between H+ and Ca2+ for the Ca2+-binding pocket33, we hypothesized that TMEM16A could be gated by Vm-dependent protonation of Glu and Asp residues within the pocket. This is indeed what we found. Our data do not rule out the presence of voltage-sensing domains, instead reveals an unexpected source of Vm dependence, namely Vm dependent protonation. As we acidified the cytosolic side of TMEM16A a large outward ICl,Vm that lacked time dependence was activated by depolarizations in the absence of intracellular Ca2+. The equilibrium constant of protonation and the apparent open probability both increased in a Vm-dependent manner. These effects were reduced after mutating Glu and Asp residues from the Ca2+-binding pocket, confirming that H+ titrate these residues. Nevertheless, in TMEM16A-5M a channel with all the residues mutated, intracellular acidification enhanced channel activity although the effect was Vm-independent. The estimated pKa of this secondary activation is between 4.5 and 6 suggesting protonation of His and probably Asp residues located in the cytosolic side. Together, our data is consistent with a mechanism depicted by the Scheme in Fig. TMEM16A gating in the absence of intracellular Ca+ ions interact in a Vm-independent manner (K1 = β1/α1) with acidic residues (shown in red) located in the cytosolic side of TMEM16A  outside the electrical field. A depolarizing stimulus will push H+ into the electrical field where they can interact with the acidic residues of the Ca2+-binding pocket (K2 = β2/α2). Once these residues are protonated (shown in navy), the channels reach the conductive state through a Vm dependent transition (K3 = β3/α3) and generate ICl,Vm.We propose that intracellular H+ observed on TMEM16A are comparable to those reported on human SLO1 BK potassium channel39. Both channels are activated in the absence of intracellular Ca2+ by intracellular acidification and the targets are Ca2+ sensing residues. Interestingly, intracellular H+ target two His residues, as well as one Asp residue located within the RCK1 domain of BK channels; mutating the His residues, abolished the activating effect of H+. In agreement with this, the equilibrium constant of BK protonation has a value of about 6.5 at +100 mV and displays shallow Vm dependence39. Here intracellular H+ activates TMEM16A by Vm-dependent and independent mechanisms. The Vm-independent pKa has a value of about 5.0 at +100 mV, which may suggest titration of Glu, Cys or His residues40 just like in BK channels.The effects of intracellular H2+ is achieved by neutralization of the electrostatic potential generated by the acidic residues of the Ca2+-binding pocket and the subsequent movement of TM6 towards TM830. This process seems to be assisted by phosphatidylinositol 4,5 bisphosphate43. Neutralization of the electrostatic potential by Ca2+ is illustrated in TMEM16A Gly644Pro mutant channels where intracellular Ca2+ can abolish the strong outward rectification displayed by these channels30. In the “constitutively protonated” TMEM16A 5 M channel the electrostatic potential has been neutralized, however, the channel was still activated by Vm and showed outward rectification. This implies that neutralizing the electrostatic potential is not sufficient to abolish rectification. This idea is supported by the strong outward rectification observed under intracellular acidic conditions, which should partially or abolish the electrostatic potential. Alternatively, TM6 may remain bound to TM4 in TMEM16A 5 M thus inducing rectification.Full activation of TMEM16A by intracellular Ca44 shows that TMEM16A is the target of extra- and intracellular H+. In both cases, protonation of acidic residues enhanced the open probability of the channel albeit extracellular H+ do so independently of Vm and Ca2+. What would be the physiological consequences of TMEM16A regulation by protons? Although we cannot answer this question yet, we envision that this regulatory process would be important to both TMEM16 channels and scramblases since the residues targeted by H+ are present in these proteins. Cancer cells overexpress TMEM16A channels and experience a large pH gradient46, conditions that facilitate cell migration and cancer progression. In these cells, the extracellular side is acidic whereas the cytosol is alkaline, and this favours TMEM16A activity. A key salient property of this regulation is that increasing the [H+]e or the [H+]i increases the current size without changing the fast kinetics. The activation of TMEM16A by Vm in the absence of intracellular Ca2+ occurred in less than 1.0 ms, well within the time scale of the electrical activity of excitable cells. Rapid activation of TMEM16A can regulate the electrical activity by inducing membrane depolarization or by accelerating action potential repolarization; this has been shown in neurons from dorsal root ganglia, cholinergic neurons of the medial habenula and muscle cells47. Thus, activation of TMEM16A enables neurons to respond to thermal stimulus, control anxiety-related behaviour47 and increase the frequency of action potentials in skeletal muscle cells of zebrafish48. A more direct physiological role for TMEM16A regulation by protons is suggested by its simultaneous activation with H+ ATPase in the apical membrane of proximal tubules of mouse kidney49. In this scenario, a parallel Cl− flux via TMEM16A would serve as a counter ion for H+ transport by the V-ATPase. Also, it is interesting to notice that the dimeric channels TMEM16A and CLC-0 are both activated by intracellular to protons50. In conclusion, we propose that intracellular H+ endow TMEM16A with Vm gating in the absence of intracellular Ca2+ by a mechanism that includes Vm-independent titration of cytosolic residues and Vm-dependent titration of acidic residues located in the Ca2+-binding pocket.The present work together with a previous report from our group2/5% CO2 atmosphere. Wild type mouse TMEM16A (ac) and mutant DNAs were cloned into pIRES2-EGFP  or pEGFP-N1 vectors. Mutations were introduced using the Quick-change kit (Aligent) and verified by sequencing. HEK-293 cells were transfected with 1 µg/µl cDNA using Polyfect transfection reagent (QIAGEN), according to the manufacturer’s instructions. Cells were used 12 h after transfection. For whole cell recordings, we seeded cells at low density whereas for inside-out recordings stably transfected cells with TMEM16A or transiently transfected mutants were plated onto poly-l-lysine coated coverslips.Human embryonic kidney 293 cells (HEK-293) were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% FBS and 0.1% penicillin-streptomycin at 37 C° in a 95% O−) currents  were recorded at room temperature (21–23 °C) from whole cells or inside-out patches expressing wild type (WT) or mutant TMEM16A channels using the patch clamp technique as we previously reported51. We selected EGFP fluorescent HEK-293 cells using an inverted microscope equipped with UV illumination. Borosilicate patch pipettes were fabricated using P-97 electrode puller (Sutter Instruments CO.). The electrode resistance was 3–5 MΩ for whole cell or 1–2 MΩ for inside-out patches. The stimulation protocol consisted of Vm steps from −100 to +160 mV delivered every 7 s from a holding potential of −30 mV, followed by a repolarization potential to −100 or +100 mV. ICl,Vm from inside-out patches were recorded using a 635 ms Vm ramp (−100 and +200 mV). Data were acquired using an Axopatch 200B amplifier and the pClamp10 software (Molecular Devices). ICl,Vm was filtered at 5 kHz and digitized at 10 kHz while the bath was grounded using 3 M KCl agar-bridge connected to an Ag/AgCl reference electrode. Solutions were applied using a home-made gravity perfusion system.Vm-activated macroscopic chloride  by adding D-mannitol to preclude activation of endogenous volume-sensitive Cl− currents52. To determine the anion selectivity sequence, we replaced 100% Cl− with the desired anion. We constructed concentration-response curves for the blockade of ICl,Vm by tannic and anthracene-9-carboxylic acids by perifusing standard external solution containing increasing concentrations of the inhibitors. To avoid precipitation of tannic acid, we used an extracellular solution containing 139 mM NaCl instead of 139 mM TEA-Cl.The extracellular solution contained (in mM): 139 TEA-Cl, 20 HEPES, 0.5 CaCl2+] = 0 and pH = 7.3 contained (in mM): 40 TEA-Cl, 50 HEPES, EGTA-TEA 25.24 and 85 D-mannitol. To obtain [Ca2+] = 0.2 µM, we added 5.24 mM CaCl2 to the standard intracellular solution and TEA-Cl was reduced to 30 mM. To test the effect of intracellular [H+]i on ICl,Vm, the pH of the standard solution was adjusted to 4.0 with 50 mM tartaric acid; to 4.0, 5.0, and 6.0 with 50 mM MES; to 7.3 with 50 mM HEPES; to 8.0 and 9.0 with 50 mM bicine. The pH was adjusted with TEA-OH, the tonicity was 290–300 mosm/kg and the free [Ca2+] was calculated using the MAXCHELATOR program (maxchelator.stanford.edu).The standard intracellular solution with i on ICl,Vm was quantified from concentration-response curves using the Hill Eq. N is the number of H+ or B interacting with one channel according to the following Scheme I:max and Imin are the maximum and minimum response, IC50 is the concentration of inhibitor ([B]) needed to obtain half Imax − Imin inhibition; it also represents the equilibrium constant of protonation K, and N is the Hill coefficient. For the Vm dependence of K, we fitted titration curves at different Vm, the corresponding K was converted to pK (−log K) and plotted against Vm. The curve was fit with Eq. 55:pK0 is the effective pHi needed to obtain half-activation when Vm = 0 mV, R is the gas constant, T is absolute temperature, F is Faraday’s constant, z is the charge, δ is the electrical distance from the inside. To obtain the V0.5 value of TMEM16A-WT, the current-voltage curve was adjusted to the following equation:0.5 is the voltage at which the 50% of channels were activated, Gmax is the estimated maximum conductance and dx is the Vm sensitivity. We estimate the liquid junction potentials using the Clampex routine of pClamp and used them to correct the reversal potential values.The effect of different blocker concentrations ([B]) or [Hentclass1pt{minimaPooled data are presented as mean ± S.E.M. of n (number of independent experiments). Statistically significant differences between means were determined using a Student t-test or ANOVA.Supplementary Information.


Here, we outline the OnGuard modelling environment and its use as a guide to predicting the emergent properties arising from the interactions between non-linear transport processes. We summarise some of the recent insights arising from OnGuard, demonstrate its utility in interpreting stomatal behaviour, and suggest ways in which the OnGuard environment may facilitate ‘reverse-engineering’ of stomata to improve water use efficiency and carbon assimilation.Plant membrane transport, like transport across all eukaryotic membranes, is highly non-linear and leads to interactions with characteristics so complex that they defy intuitive understanding. The physiological behaviour of stomatal guard cells is a case in point in which, for example, mutations expected to influence stomatal closing have profound effects on stomatal opening and manipulating transport across the vacuolar membrane affects the plasma membrane. Quantitative mathematical modelling is an essential tool in these circumstances, both to integrate the knowledge of each transport process and to understand the consequences of their manipulation  Gas exchange is regulated by exogenous signals, such as CO2, water availability, and light  ([Ca2+]i) and pH, and in many cases by reactive oxygen species (ROS), and protein phosphorylation ; furthermore, we can describe these dependencies in quantitative terms i inactivates the inward-rectifying K+ channels of the guard cell i and K+ channel activity, and we can safely place the mechanistic details in a mathematical description that subsumes the intermediate kinetics. In effect, such a phenomenological approach introduces modules with adjustable levels of resolution that may be expanded if, and when, studies come to focus on a specific module i and cytosolic pH i and pH — depend on interactions between the transporters, cytosolic and vacuolar buffering for pH and [Ca2+]I, and metabolism, just as they do in vivo. So, in general, the task of interpretation reduces to one of tracing the sequence of events following a trigger, or change in a specific model parameter, through the network of interrelated homeostatic processes. Here, the output variables, their kinetics, flux, and metabolic origins are most useful and will help in identifying emergent behaviours of the system that are often the most informative aspects of any modelling effort.Interpreting the output of an OnGuard2 simulation requires the user to interrogate the model variables. Changes in each of these variables — for example, solute concentration, the associated rates of ion and solute flux through each of the transporters, the membrane voltages, i leads to plasma membrane hyperpolarisation, as pump activity is no longer subject to this feedback control. Modelling shows that the enhanced H+-ATPase activity elevates pHi and, by hyperpolarising the membrane, it also promotes Ca2+ influx through the inward-rectifying Ca2+ channels to elevate [Ca2+]i. The changes in [Ca2+]i and pHi, in turn, suppress the inward-rectifying K+ channels while the elevated pHi enhances the outward-rectifying K+ channels. These predictions are borne out experimentally and demonstrate an unexpected set of connections between K+ channel and H+-ATPase activity i. Again, the changes in pHi and [Ca2+]i are a direct consequence of full H+-ATPase activity and its insensitivity to Ca2+, which together promote cytosolic alkalinisation unchecked by the concurrent Ca2+ influx through the inward-rectifying Ca2+ channels and [Ca2+]i elevation, in this case also in the dark. Both the pHi and [Ca2+]i signals also suppress the inward-rectifying K+ channels and the alkaline pHi enhances the outward-rectifying K+ channels [ost2 mutants [Finally, eliminating the sensitivities to both light and Cachannels . These c mutants ,8.2, and light, but without connection to guard cell mechanics. We expect the outcomes of modelling strategies, such as those we have outlined with OnGuard2, will prove central to guiding research and its application in the future.Quantitative computational modelling is a gold standard for physiological analysis. These tools enable researchers to explore emergent properties associated with membrane transport and cellular signal processing. For stomata, the OnGuard platform encapsulates the mechanics of stomatal behaviour across scales from the molecular events of channel gating through to whole-plant transpiration. From an ecophysiological perspective, it bridges scales in a way that demonstrates the real potential for ‘reverse-engineering’ of efficiencies in whole-plant water use and carbon assimilation. The mechanics of membrane transport and metabolism in guard cells are marked by an extraordinary wealth of knowledge and quantitative kinetic detail at the cellular level that underpin their successful modelling. This knowledge can now be expanded to the challenges of foliar transpiration and carbon capture that previously were described empirically through quasi-linear relations with atmospheric humidity, COThe transport of osmotically active solutes across the membranes of stomatal guard cells drives stomatal movements, thereby regulating gas exchange for photosynthesis while protecting the leaf from excessive water loss via transpiration. The complexity of this transport makes mathematical modelling an essential tool, especially for understanding transport and its interactions that regulate gas exchange.Modelling strategies have commonly focused either on the molecular mechanics of transport and the associated metabolic processes or on the macroscopic properties of stomatal gas exchange in the leaf. Very few modelling efforts have sought to bridge these scales to interlock the molecular events in the guard cell to whole-plant gas exchange in the field, and only one platform incorporates the wealth of molecular kinetic detail needed to accurately describe guard cell membrane transport with true predictive power.Looking ahead toward future efforts to engineer crops for enhanced efficiencies in photosynthesis and water use, there is a clear need to bridge the molecular-macroscopic scales within a single, computational framework that is capable of spanning metabolic events including that of carbon assimilation.


AITC (200 μM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 μM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca Human cardiac fibroblast, Transient receptor potential ankyrin 1, TRPA1, Allyl isothiocyanate, Methylglyoxal, Nonselective cation channels In the presence of extracellular Ca2+, the application of AITC (200 μM) induced a rapid increase of [Ca2+]i and subsequently decreased to a steady-state level . In contrast, AITC did not affect [Ca2+]i in the absence of extracellular Ca2+ , an agent known to block various subtypes of TRP channels [2+]i rise  increased the inward current at a holding potential of -40 mV  on membrane currents in hCFs. The cells were held at -40 mV. The patch pipette was filled with the Csf -40 mV A. The cu current C. Furthe current D. Effect current A. The I–2+]i in hCFs i that is blocked by HC030031 or by siRNA-induced knockdown of TRPA1 i in the absence of extracellular Ca2+, but in the presence of extracellular Ca2+, AITC increased [Ca2+]i. The AITC-induced [Ca2+]i was blocked by RR or La3+ in a similar manner as AITC-induced nonselective cation currents. These results provide the direct evidence for functional existence of TRPA1 channels in hCFs, by using [Ca2+]i measurements and the patch-clamp techniques. However, it remains unclarified whether basal activity of TRPA1 channels without agonists can contribute to basal [Ca2+]i. RR failed to affect basal [Ca2+]i and background current, proposing that TRPA1 channels are less activated during basal conditions, compared with agonist stimulation. This is somewhat compatible with the following results. Andrei et al. [2+]i in cardiomyocytes, but not cardiomyocytes prepared from Trpa1−/− mice. However, Bodkin et al. [Trpa1−/− mice, suggesting that TRPA1 channels in basal state do not modify cardiac function. In the present study, La3+ further inhibited the background current with Er of approximately +0 mV, suggesting that hCFs have background nonselective cation channels other than TRPA1 as previously reported in various types of cells [TRPA1 has been reported as a highly Ca-permeable nonselective cation channel , 40 withet al.  showed si et al. , 7 reporof cells , 41, 42.2+ entry plays an essential role in fibroblasts' biological functions [+ and Ca2+ pass the TRPA1 channel, resulting in membrane depolarization and action potential generation [Several lines of evidence have shown that Caunctions , 43. Preunctions , 44, 45.unctions , 47. Becneration . Thus, tneration , 49. Furneration , and planeration . Wang etneration  also shoneration . The fur3+, a non-specific blocker, and the further studies using a more specific TRPA1 blocker and single channel recording techniques as well as freshly isolated hCFs are required to clarify the identification of TRPA1 channels in hCFs.We should acknowledge several limitations. We had used the commercially cultured healthy hCFs in this experiment. Ion channels, receptor function and calcium regulation mechanisms in hCFs may be changed after the isolation and during the culture conditions. Furthermore, we used RR and La2+-permeable nonselective cation currents activated by AITC and MG, possibly via TRPA1 in hCFs.The present results provide the first evidence for existence of functional CaT. Nakajima: Performed the experiments; Wrote the paper.G. Oguri, H. Kikuchi and S. Obi: Conceived and designed the experiments; Performed the experiments.F. Nakamura and I. Komuro: Contributed reagents, materials, analysis tools or data.10.13039/501100001691JSPS KAKENHI (18K08084) and T. Nakajima was supported by JSPS KAKENHI (19H03981).S. Obi was supported by Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca After 36–48 h, VMs were perfused with Tyrode’s solution containing 1 mmol/L Ca2+. Isoproterenol  was added to the solution to stimulate β-adrenergic receptors. Low-dose caffeine (200 µmol/L) was added to induce RyR2 leak. Myocytes were preincubated with specific mitochondrial superoxide scavenger mitoTEMPO  for 10 min prior to experimentation, and was included in the perfusion solution. Biosensor G-CEPIA1er was excited using 488 nm line of argon laser and fluorescence emission was collected at 500–550 nm, measured in x–y mode at 400 Hz sampling rate. Resting VMs were exposed to sarco/endoplasmic reticulum Ca2+-ATPase (SERCa2a) inhibitor thapsigargin (10 μmol/L) after 5 min in ISO or ISO and caffeine, and fluorescence signal from G-CEPIA1er was monitored using confocal microscopy. The time constant of decay of G-CEPIA1er was used as a measure of the leak by fitting fluorescence data to a monoexponential function m = Kd ×  (F – Fmin)/(Fmax – F), where Kd of mtRCamp1h = 1.3 µmol/L, fluorescence was converted to [Ca2+]m for each myocyte. Analysis parameters included peak mtRCamp1h [Ca2+]m (μmol/L), the time to peak amplitude (s), the first derivative of [Ca2+]m [2+ s−1) and the rate of decay (s−1).To assess mito-Ca [Ca2+]m , 65  at room temperature for 10 min in Ca2+-free Tyrode’s solution, followed by a 8 min wash in Tyrode’s solution containing 1 mmol/L Ca2+. Myocytes were perfused with Tyrode’s solution containing 1 mmol/L Ca2+ at room temperature during recordings. Fluo-3 AM was excited at 488 nm and fluorescence emission was collected at 500–550 nm wavelengths in line scan mode at 200 Hz sampling rate. The pacing protocol was followed as described above, with pacing for 5 min at 2 Hz, followed by the addition of either ISO (50 nmol/L) or ISO and low-dose caffeine (200 µmol/L), then further pacing for 5 min. For experiments with mitoTEMPO, VMs were preincubated with mitoTEMPO  for 10 min prior to experimentation, and mitoTEMPO was included in the perfusion solution. Line scans were recorded during the last minute of pacing. Cytosolic Ca2+ transient amplitude is presented as ΔF/ΔF0, where F0 is basal fluorescence and ΔF = F–F0.To measure cytosolic Ca2+ transient, intact VMs were loaded with Fluo-3 AM (Invitrogen) at room temperature for 20 min in Tyrode’s solution containing 0.5 mmol/L Ca2+, followed by a 20 min wash in Tyrode’s solution containing 1 mmol/L Ca2+. Myocytes were then perfused with Tyrode’s solution containing 2 mmol/L Ca2+ and ISO (100 nmol/L) at room temperature during Ca2+ transient recordings. For experiments with mitoTEMPO, VMs were preincubated with mitoTEMPO  for 20 min prior to experimentation, and mitoTEMPO was included in the perfusion solution. Fluo-3 AM was excited at 488 nm and fluorescence emission was collected at 500–550 nm wavelengths in line scan mode at 200 Hz sampling rate. To test for the propensity of triggered activity, VMs were stimulated for 10 s at 0.5 Hz and the latency between the last pacing stimulus and the subsequent SCW was calculated. To assess SR Ca2+ load, high-dose caffeine (10 mmol/L) was applied at the end of the experiments. The data are presented as ΔF/F0, where F0 is the basal fluorescence and ΔF = F–F0.To measure intracellular Ca2+ using MitoSOX Red mitochondrial superoxide indicator . The indicator was excited with 514 nm line of an argon laser and emission was collected at 560–660 nm, measured in the x–y mode. Fluorescence of MitoSOX was normalized to the maximum fluorescence signal obtained by application of DTDP (200 µmol/L).The emission of mitochondrial superoxide was measured in isolated VMs in Tyrode’s solution containing 2 mmol/L Ca2 for 36–48 h. Myocytes were then prepared for immunofluorescence by fixing with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100/PBS (pH 7.2) containing 1% BSA. Samples were then probed using RyR2 primary antibody. Secondary antibody used was goat anti‐mouse IgG (H + L) cross‐adsorbed secondary antibody Alexa Fluor 633.Cultured control rat VMs plated on laminin-coated glass coverslips were infected with G-CEPIAer or ERroGFP_iE adenoviruses and were cultured at 37 °C in 95% air and 5% CO2 for 36–48 h. Live VMs were then stained with MitoTracker Deep Red FM  for 15 min, then washed in Tyrode’s solution prior to imaging. The indicator was excited with 546 nm line of an argon laser and emission was collected at 550–660 nm, measured in the x–y mode.Cultured control rat VMs plated on laminin-coated glass coverslips were infected with OMM-HyPer adenovirus and were cultured at 37 °C in 95% air and 5% COFreshly isolated rat VMs were treated with isoproterenol (50 nmol/L),and caffeine (200 μmol/L) for 4 min prior to 2 Hz pacing for 1 min at room temperature. Cells were then immediately lysed in lysis buffer from Cell Signaling (Cat#9803S), supplemented with phosphatase  and protease inhibitor cocktails  as described previously .RyR2 was immunoprecipitated from cell lysate using anti-RyR2 antibody (5 μL) in 0.5 mL RIPA buffer overnight at 4 °C. Samples were incubated with Protein A/G Plus-agarose beads (Santa Cruz cat # sc-2003) for 1 h at 4 °C and washed three times with RIPA buffer.To determine the oxidation status of RyR2, the Oxidized Protein Western Blot Kit was used, whereby carbonyl groups of immunoprecipitated RyR2 were derivatized to 2,4 dinitrophenylhydrazone (DNP) by reaction with 2,4 dinitrophenylhydrazine. For control, we used the kit-provided Derivatization Control Solution. The DNP-RyR2 protein samples were separated on 4–20% Mini-PROTEAN TGX gels  and DNP-associated signal was assessed by the kit-provided anti-DNP rabbit primary antibody and anti-RyR2, followed by HRP-conjugated anti-rabbit goat secondary antibody and anti-mouse lgG(H + L),HRP secondary antibody.Blots were developed with ECL (Bio-Rad Laboratories) and quantified using Image J  and Origin 8 software.2+-free Tyrode’s solution before mounting on a Langendorff apparatus. Hearts were then perfused with Tyrode’s solution containing 2 mmol/L Ca2+ for 15 min. The atria were removed and ventricles were homogenized using Tissue Tearer Model 985,370 in 1 mL of buffer containing: Tris–HCL , NaCl (150 mmol/L) NaF (5 mmol/L), Na3VO4 (1 mmol/L), 0.5% Triton-X 100, protease inhibitor and phosphatase inhibitor. The homogenate was centrifuged at 12000xg for 15 min at 4 °C. RyR2 was immunoprecipitated from the supernatant (500 mL) using anti-RyR2 antibody (3 µL) for 2 h at 4 °C. The samples were then incubated with Protein A/G Plus-agarose beads  for 1 h at 4 °C and washed three times with RIPA buffer.Hearts were rapidly excised from isoflurane-anesthetized mice and immersed in cold CaTo determine the oxidation status of RyR2, the Oxidized Protein Western Blot Kit was used, whereby the carbonyl groups of immunoprecipitated RyR2 were derivatized to DNP by reaction with 2,4dinitrophenylhydrazine. Kit-provided Derivatization Control Solution was used as control. The DNP-RyR2 protein samples were separated on 4–20% Mini-PROTEAN TGX gels and DNP-associated signal was assessed by the kit-provided anti-DNP rabbit primary antibody and anti-RyR2, followed by HRP-conjugated anti-rabbit goat secondary antibody and anti-mouse lgG (H + L), HRP secondary antibody.Blots were developed with ECL (Bio-Rad Laboratories) and quantified using Image J , supplemented with phosphatase  and protease inhibitor cocktails  as described previously . Samplesn(N) = number of animals, and lowercase n = number of VMs. Statistical significance between groups was calculated using Student’s t test (paired and unpaired), Fisher’s exact test and one-way ANOVA with Bonferroni post hoc test, where appropriate. A p value of less than 0.05 was considered significant.All statistical analysis was performed using Origin 8 (OriginLab). Data are presented as mean ± standard error (SEM). Uppercase When used at low concentrations, caffeine acts as an agonist of RyR2. We first sought to demonstrate that low-dose caffeine (200 µmol/L) could reproduce the leaky SR phenotype observed in cardiac disease, using cultured VMs isolated from healthy rat hearts.2+]SR, we utilized genetic probe G-CEPIAer, a GFP-based biosensor spatially restricted to the SR, with a Kd ~ 672 µmol/L for Ca2+ m) in rat VMs  was used to obtain maximal fluorescence of saponin-permeablized VMs.We next investigated whether Cafor Ca2+ , we examVMs Fig. . After iVMs Fig. a. Probe 2+ leak. After this protocol, VMs were saponin permeabilized. Minimal  and maximal  fluorescence signal was obtained, allowing for recalculation of signal to [Ca2+]m. As shown in Fig. 2+]m increases during pacing, with significantly reduced peak [Ca2+]m when treated with ISO and low-dose caffeine, compared to ISO alone  into the mitochondria during pacing. The rate of transient decay was significantly increased after caffeine treatment . Images in Fig. 2+]m reduction under conditions of enhanced RyR2 activity.Using cultured rat VMs stained with mitochondrial voltage-sensitive dye TMRM (20 μmol/L for < 1 min), we assessed whether increased RyR2 activity modifies mitochondrial membrane potential , subjecting VMs to a burst-pace pause protocol . Representative images of Ca2+ transients and Fluo-3 AM fluorescence profiles (ΔF/F0) are shown in Fig. 2+ transient amplitude did not show significant differences between CPVT and WT VMs . This significantly increased SCW latency and restored SR Ca2+ content to near WT levels, suggesting that ROS scavenging can partially stabilize proarrhythmic RyR2 activity in CPVT VMs, similar to the effects of mitoTEMPO in rat VMS acutely challenged with caffeine  may also play a role in reducing net [Ca2+]m during enhanced SR Ca2+ leak, as seen in conditions of cytosolic Na+ overload m with a genetically targeted biosensor in live VMs do not reflect total mito-Ca2+ accumulation, because we are not tracking Ca2+ complexed with phosphates. However, according to Chalmers and Nicholls [2+ must first be increased to promote the formation of Ca2+-phosphate complexes. A more likely scenario is that differences in cellular [Na+] between control and diseased VMs, and thereby changes in NCLX-mediated mito-Ca2+ efflux, are omitted upon mitochondria isolation. Furthermore, the reduction of cytosolic Ca2+ transient amplitude and [Ca2+] available for uptake observed in live VMs are not directly mimicked in in vitro studies. At the same time, our data demonstrating an increase in maximal mito-Ca2+ uptake rate (Fig. 2+] in isolated mitochondria from samples with leaky RyR2s. Future studies using protocols that match mito-Ca2+ fluxes in live cells and isolated preparations are needed to reconcile this controversy.Importantly, our results contradict the findings obtained in several models with hyperactive RyR2 channels. The Camutation  and in amutation . An explNicholls , 54, fre2+-dependent process m and ΔΨm may produce secondary effects on other key components of intracellular Ca2+ handling machinery including SERCa2a, Na+/Ca2+ exchanger and L-type Ca2+ channels [2+ leak is the major contributor to defective Ca2+ homeostasis in conditions with hyperactive RyR2s.Sarcoplasmic reticulum Cachannels , 33, 68.channels –6, 18. T2+ overload. However, under conditions mimicking stress, these mechanisms fail to fully alleviate mito-Ca2+-dependent increase in mito-ROS, resulting in oxidation of RyR2. This further increases channel activity and exacerbates defective intracellular Ca2+ homeostasis.Taken together, our results suggest that both acute and inherited increase in activity of RyR2 evoke multiple mechanisms that protect mitochondria from Ca


Ca2+ is transported through specific channels and transporters in the plasma membrane and subcellular organelles such as the endoplasmic reticulum and mitochondria. Therefore, dysregulation of intracellular Ca2+ homeostasis may lead to cell dysfunction and disease. Accordingly, chemical compounds from natural origin and/or synthesis targeting directly or indirectly these channels and proteins may be of interest for the treatment of cell dysfunction and disease. In this review, we show an overview of a group of marine drugs that, from the structural point of view, contain one or various heterocyclic units in their core structure, and from the biological side, they have a direct influence on the transport of calcium in the cell. The marine compounds covered in this review are divided into three groups, which correspond with their direct biological activity, such as compounds with a direct influence in the calcium channel, compounds with a direct effect on the cytoskeleton and drugs with an effect on cancer cell proliferation. For each target, we describe its bioactive properties and synthetic approaches. The wide variety of chemical structures compiled in this review and their significant medical properties may attract the attention of many different researchers.Intracellular Ca A variety of heterocyclic compounds modulate different biological functions. Here we will focus on heterocyclic compounds that affect intracellular calcium signal through their effect on ion channels, cytoskeleton and impact on tumor cell proliferation.2+ is a fundamental ion in cellular metabolism as well as participating in the construction and maintenance of bones and teeth. Furthermore, ionic intracellular Ca2+ is also critically involved in cell signaling, as this ion is considered one of the most important second messengers. Thus, intracellular Ca2+ ions modulate a large number of cell functions from exocytosis and muscle contraction to regulation of gene expression, cell proliferation and cell death  is similar to the cytosol and can only be increased temporarily after stimulation . Si. Si61, w2 reductive cyclization. More recently, in 2017, Saito and Nakata synthesized the enantiomer of the ZA′B′C′D′-ring system of maitotoxin through a convergent strategy [Other groups have also made great efforts in the synthesis of different parts of this amazing molecule. In 2008, Nakata published a series of papers describing the synthesis of C’D’E’F’ , WXYZA’ strategy .65 is a polyether toxin related to ciguatoxins and produced by different dinoflagellates, particularly Lingulodinium polyedrum and Gonyaulax spinifera  cells, and this effect was blocked by external Ca2+ chelation and nifedipine, thus suggesting that YTX-induced apoptosis is mediated by activating a Ca2+ entry pathway and Ca2+ overload [Yessotoxin (YTX) pinifera . This to neurons . Ca2+ enchannels . Yessotooverload .67, obtained by ruthenium oxidation of intermediate alkyne 66, is treated with TsOH to produce tetracyclic dihemiketal 68. O-methylation of the two hemiketals hydroxyl groups 68 and reductive etherification results in the formation of BCDE ring system 69 of yessotoxin  and the formation of a cyclic acetal from a hydroxy ketone (83 to 84) [In 2005, Oishi reported a route to the FGHIJ fragment 84 , and in 84 . The mai3 to 84)   is a highly thermostable, polyhydroxylated and partially unsaturated compound with as much as eight double bonds within a long carbon chain. It is produced by  ouabain . As a co ouabain .PTX also elicits the release of glutamate from cerebellar granule cells leading to their depletion as well as prostaglandins from rat aorta with intact endothelium . Finally94 was elucidated, as depicted in Initial studies of palytoxin focused on determining its stereochemistry, relying on the synthesis of some of its degradation products . Thus, t95. Palytoxin carboxylic acid retrosynthesis results in a division in eight fragments: C1-C7 (96), C8-C22 (97), C23-C37 (98), C38-C51 (99), C52-C75 (100), C76-C84 (101), C85-C98 (102), C99-C115 (103).Seven years after its isolation, Kishi reported its first total synthesis . He focuBefore proceeding to the coupling of the fragments, they needed to be synthesized. Each segment requires a different coupling, being some of them relatively straightforward, while others are more challenging.104).Coupling between C22-C23 and C37-C38 goes via Wittig reaction followed by hydrogenation, so a C8-C51 chain is synthesized /Cr(II)-catalyzed coupling reaction was then used to bond obtained .102 and 103 are coupled by a Wittig reaction producing the C98-C99 olefinic bound. 106 reaction with 101, relying on Ni(II)/Cr(II) catalysis, followed by PDC oxidation and Wittig olefination, produces C84-C85 olefinic bond . As numerous protection groups have been used, a deprotection step is required to obtain the final product. All deprotection steps produce a 30% overall yield of the carboxylic acid 95  provides palytoxin 94 [Once the initial eight fragments have been coupled to form  acid 95 . FinallyAfter this first total synthesis, some other approaches have been developed, being the main objective of most of them the quantitative stereospecific synthesis of a fragment. For instance, Still’s approach pursues the synthesis of C30-C43 fragments by a stereospecific macrocyclically controlled remote asymmetric induction process . On the 2+ channels, specifically intracellular Ca2+ channels, as the inositol trisphosphate (IP3) receptor, a highly relevant, ligand-gated Ca2+ channel located in the membrane of the ER and responsible for agonist-induced Ca2+ release from intracellular stores. Accordingly, latruculin A may induce Ca2+ release from the ER, then the depletion of intracellular Ca2+ stores may activate store-operated Ca2+ channels and secondary Ca2+-dependent Na+ entry, which may promote membrane depolarization. These effects are similar to those induced by cytochalasin D, a well-known actin disruptor. Latruculin A-elicited depolarization consisted of a positive shift in Vm, which reached the threshold of activation of voltage-dependent Ca2+ channels, thus triggering an action potential. In Ca2+-free medium latruculin A lacked the ability to induce action potentials, while it was abolished upon removal of external Na+. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine [2+ waves that depend on an intact cytoskeleton [Latrunculin A is a member of the family of natural products and toxins termed latrunculins. At low μM concentrations, it binds actin monomers preventing their polymerization, thus resulting in the disruption of actin filaments of the cytoskeleton . This efyanodine . Actin pyanodine . Anotherskeleton .Latrunculia magnifica (Keller) [Latrunculins A and B  were fir(Keller) .111 with aqueous HCl deprotected the TBS group and also affected the cyclization into hemiacetal, which was methylated with catalytic CSA and methanol to give 112. Ring-closing alkyne metathesis (RCAM) of intermediate 113 (obtained from 112 in four steps) produces the highly strained 16-membered cycle 114 in an enyne-yne metathesis catalyzed by a molybdenum complex. Compound 114, the smallest ring size that has been synthesized by an enyne-yne metathesis, forms 109 in three further steps in 41% overall yield [Fürstner and coworkers designed a versatile synthetic route to different members of the latrunculin family through ring-closing enyne-yne metathesis ,89,90. TE,Z-diene formed by Wittig reaction of an E-ylide with an aldehyde [As latrunculin A differs from other members of its family in the presence of a conjugated diene, White’s convergent synthetic approach proposes the formation of an acyclic aldehyde .115 with E-ylide 116 gives E,Z-diene 117, which is further elaborated to produce intermediate aldol 118 in five steps. The final steps to 109 from 118 are quite straightforward. Treatment with HF and methanol allows selective deprotection of SEM group and subsequent cyclization, thus closing the desired tetrahydropyran ring through the formation of a methyl ketal. Finally, 119 is transformed to 109 in three additional steps , where the longest linear sequence is 17 steps [120 with n-Bu2BOTf and i-Pr2NEt followed by reaction with aldehyde 121 produces 122 (major isomer), which is then transformed to 123 in 22% yield over seven steps. Wittig coupling of aldehyde 123 with ylide 124 furnishes a cis-trans diene 125 that in four steps produces 109  methanol. Then, ester cleavage to 128, followed by Mitsunobu reaction, gives macrolactone 129. Simple hydrolysis with acetic acid affords the desired latrunculin A 109  2,6-drofuran . Altmann133 was prepared by a Prins cyclization reaction of the homoallylic alcohol 131 and the acetal 132 4 and the Fmoc group (piperidine/DMF) and macrocyclization  yielded the desired patellamide A 138 -catalyzed (3 + 2) annulation strategy . The totin D 151 .162 and other analogs in 2019 [158 with β-bromo-β-nitrostyrene 157, giving the central 1,2,4-trisubstituted pyrrole core in a highly regioselective manner. Coupling of pyrrole-2-carboxylic acid 159 with phenol 160, followed by Pd-catalyzed intramolecular oxidative C-H arylation, afforded the pentacyclic 161, lamellarin G trimethyl ether, in 44% yield. Lamellarin D trimethyl ether 162 was obtained by dehydrogenation with DDQ in excellent yield . Conventional heating of enaminone 165 in ethyl bromoacetate provided a tricyclic product, which was oxidated by DDQ to give the unsaturated ring 166. A final saponification/lactonization process furnished lamellarin D trimethyl ether 162 in yields consistently above 96% (More recently, Michael and coworkers have published the synthesis of lamellarin D trimethyl ether rocycles . Eschenmbove 96% . OverallIn this review, we have tried to show an overview of a group of marine drugs that, from the structural point of view, contain one or various heterocyclic units in their core structure and, from the biological side, have a direct influence on the transport of calcium in the cell. The marine compounds covered in this review are divided into three groups, which correspond to their direct biological activity, such as compounds with a direct influence in the calcium channel, compounds with a direct effect on the cytoskeleton and drugs with an effect on cancer cell proliferation. For each target, we have described its bioactive properties and synthetic approaches. The wide variety of chemical structures compiled in this review and their significant medical properties will attract the attention of many researchers.