CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.  •First structure of consecutive scavenger receptor cysteine rich domains in CD6•Structure of the two N-terminal domains of CD166 which is the ligand for CD6•Mapping binding sites on CD6 and CD166•Insight into how CD6 and its interactions are perturbed by polymorphisms and mAbs Chappell et al. present structures of the T-cell surface receptor, CD6, the first of consecutive scavenger receptor cysteine rich domains and its ligand, CD166. The structures give insight into how CD6 and its interactions are perturbed by competition between homophilic and heterophilic interactions, SNPs, and mAbs. The extracellular region of CD6 contains three scavenger receptor cysteine rich (SRCR) domains and a membrane proximal stalk . CD6 engof CD166 .trans between apposing cells , were stably expressed in Lec 3.2.8.1 Chinese hamster ovary (CHO) cells , purifie1BY2, 26.7% sequence identity to CD6 domain 1, 44.5% to CD6 domain 2, and 26.1% to CD6 domain 3; X-Ray diffraction data were collected to 3.15 Å and the structure solved by molecular replacement using a search model derived from the structure of a single SRCR domain of Mac-2 binding protein  is the only residue close to the ligand binding site. We mutated CD6 S351 to S351N and compared the binding of soluble CD166 VVC to the two variants by SPR. CD166 VVC bound with a 10-fold weaker affinity to CD6 S351N compared with CD6 S351 , binding of CD166 may be sterically hindered by carbohydrate. There are seven consensus N-glycosylation sites in the three extracellular domains of CD6. In the recombinant protein, we observed electron density for GlcNAc attached to residue N229 in the second domain. There was no observable electron density for the other potential glycosylation sites in CD6. Mass spectrometry was used to determine whether recombinant CD6 S351N is glycosylated. As glycosylated peptides are not identified by liquid chromatography-tandem mass spectrometry due to the unknown mass of the N-linked carbohydrate, we quantified the precursor ion intensity obtained from extracted ion chromatograms of the FNNSNLCSsylation . The S35sylation A and S6Bsylation C and S6Dsylation .N-glycosidase F, showing that N28, N49, N229, and N351 are glycosylated and that N339 appears to be partially glycosylated . Extrapolating from the dimensions of the two CD166 IgSF V and three C domains taking the size of a C domain to be 35 Å from human CD2 domain 2 (PDB: 1HNG), the five domains in a linear array would be predicted to span ∼200 Å. This suggests there is flexibility and/or deviation from the perpendicular by CD166 between apposing cells to optimize formation of trans interactions, as has been proposed for five domain E-cadherins  SRCR domains (residues 1–364) and two V-like domains and one C-like domain (VVC) (residues 1–335) of human CD166 (UniProt: Q13740) were amplified from plasmid DNA  at 20°C. Drops were set up using 2.5 μl of a protein solution containing a complex of CD6 and CD166 at an OD280 of 4.58. Crystals grew after 4 weeks. Crystals were flash-frozen in liquid nitrogen and data were collected at the Diamond Light Source, Harwell, at 100 K on beamline i04-1.The two membrane distal immunoglobulin domains of human CD166 were crystallized using the sitting drop method in 0.1 M sodium HEPES (pH 7.5) and 25% w/v PEG-2000 MME. Drops were set up using 2 μl of a protein solution containing CD166 domains 1–3 at an OD1BY2; 3CJJ; Rwork/Rfree of 0.25/0.28 and 0.22/0.24, respectively. Coordinates were deposited in the PDB (PDB: 5a2e for CD6 and PDB: 5a2f for CD166).Native datasets were collected for CD6 and CD166, to 3.15 Å and 1.86 Å, respectively. The data were integrated and scaled using XDS  and aiml−2 mbar. The trap energy was increased to 30 V to obtain more accurate mass measurements.Purified CD6 and CD166, along with a mixture of both proteins, were subjected to native mass spectrometry analysis. Proteins were at a concentration of 20 μM and the mixture formed by incubating an equal amount of CD6 and CD166 (both at 20 μM) for 30 min at room temperature. Samples were buffer exchanged from 10 mM HEPES, 150 mM NaCl (pH 7.4) into 250 mM ammonium acetate (pH 7.6) using Biospin-6 columns (Bio-Rad). Mass spectrometry measurements were carried out on a modified Synapt G1 HDMS (Waters Corp.) Q-ToF mass spectrometer . The insP.E.C. purified and crystallized protein, solved the human CD6 and CD166 X-ray crystal structures, made the figures, and contributed to writing of the manuscript. L.I.G. purified protein and performed SPR experiments. J.Y., C.V.R., and C.M. carried out native and glycosylation mass spectrometry experiments, respectively. D.H. produced cell lines. S.M.L. and S.J. solved the human CD6 and CD166 X-ray crystal structures. M.H.B. directed the project and wrote the manuscript.


Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection.The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr Human papilloma virus (HPV) is the leading cause of cervical cancer, which is the second most common cancer in women worldwide13679111314913Each pentameric capsomer is composed of five monomers, with each monomer having five antigenic loops . Major neutralizing epitopes were found to be conformational sites that are located on these hypervariable loops1819212312Current HPV vaccine strategies focus on generating not only type-specific antibodies but also cross-type neutralizing antibodies for broader type coverage42727In this study, we characterized a human neutralizing antibody 26D1 that is specific to HPV16. The monoclonal antibody 26D1 was isolated from a memory B cell of a volunteer donor who obtained three doses of an experimental HPV16/18 VLP vaccine29in vitroA healthy female donor vaccinated with three doses of an experimental HPV16/18 L1 VLP vaccine in a Phase I clinical trial was selected for isolating HPV16-specific neutralizing antibodies (N-mAb) from individual memory B cells. As reported, antibodies cloned and expressed from singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), which could be assessed for expression, folding, and function  H and VL segments of 26D1 were assigned to IGHV4-39*01 and IGLV1-44*01 by IMGT, respectively. CDR sequences of 26D1 defined using the Kabat system50 value of 66 ng/ml  26D1 possesses a high affinity to HPV16 VLPs and potent viral neutralization activity.To characterize the human neutralizing antibody, the heavy- and light-chain immunoglobulin genes from this individual B cell well were amplified and cloned into IgG1 expression vectors, and the recombinant IgG1 antibody was named as 26D1. The V50 values of 26D1 and H16.V5 for heparan sulfate in this inhibition assay are 40 mg/ml and 100 mg/ml, respectively  for HPV16 residues on the HPV16 L1 backbone, and another set of 10 different HPV6:16 hybrid VLPs that substituted HPV16 loop residues  for HPV6 sequences on the HPV6 L1 backbone . We testThe 26D1 interacting surface loops were further verified by molecular docking simulations3637140 on one of these two DEa loops was also predicted to be involved in 26D1 binding , all except three parental Ala residues, were successively mutated to Ala, which did not impair the formation of VLPs  , left. THPV16 VLPs with DE (DE-HPV16:6 VLP mutant) and FG loops (FG-HPV16:6 VLP mutant) swapped with the type 6 loops were also used to analyze competition reactivity between 26D1 and H16.V5. For DE-HPV16:6 VLP mutant binding on the chip, the saturation reactivity of 26D1 was dramatically reduced to be 41 RU, and 76% of these 26D1 binding epitopes were blocked by H16.V5 pre-saturation (111 RU) , right. The capacity of 26D1 to block the reactivity of human sera to HPV16 VLPs was evaluated using a panel of 16 human serum samples. Eight patient samples and eight serum samples from vaccinees, who were previously determined to be seropositive in a binding assay to HPV16 VLPs, were selected at random. The biosensor method was also used for this inhibition assay. Among the eight naturally HPV-infected serum samples, 26D1 was shown to completely block the reactivity of three samples to HPV16 capsids . In cont181, Asp184 on the BC loop; Val141, Asp142 and Asn143 on the DE loop; Val267, Gln269, Asn270, Ser280, Gly281, Ser282, Thr283 and Asn285 on the FG loop), and four residues are on the HI and DE loops on the second neighboring L1 protein 138.DE, Ala139.DE, Gln181.EF, Ser282.FG, Asn285.FG, Ile348.HI, and Lys361.HI) were essential components in the common epitopes interacting with four other HPV16 N-mAbs138, Ala139, Asp142, Asn143) and three residues of the FGb loop  were overlapping epitopes targeted by both H16.V5 and 26D1, four of which  were included in the common epitopes138 and Ser282) on this patch were predicted to form a hydrogen bond in the paratope of 26D1 ; three patients were diagnosed with low-grade squamous intraepithelial lesion (LSIL), and one patient sample was diagnosed with high-grade squamous intraepithelial lesion (HSIL). All serum samples were determined to be seropositive for antibody binding activity against HPV16 VLPs.+ memory B cells were isolated from the PBMCs using the EasySep™ Human Memory B Cell Isolation Kit . To promote memory B-cell differentiation into antibody-secreting plasma cells, memory B cells were seeded at an average of 1.5 B cells/well in 96-well plates in the presence of 50 Gy irradiated CD40 ligand feeder cells and 50 ng/ml recombinant human IL-21 protein . The culture supernatants were collected 14 days later and screened for binding activity to the HPV16 VLPs and neutralizing activity in the HPV16 pseudovirus (PsVs)-based neutralization. Total RNA was isolated from positive lysed B-cell cultures using an RNeasy Micro kit , and reverse transcription was carried out using the SuperScript III First-Strand Synthesis SuperMix kit and specific IgG1 reverse primer . Heavy and light chain variable regions were then amplified by PCR using IgG1 VH, VK, Vλ family-leader region-specific primersA total of 40 ml of whole blood from the female vaccinee was collected for peripheral blood mononuclear cell (PBMC) isolation. IgGThe concentration of IgG1 in B cell culture supernatants was assessed from 2 plates randomly chosen from total of 5 plates in ELISA assay. Maxisorp Nunc-Immuno Plates  were coated with 50 μl per well of goat anti-human IgG1 (Fc) antibody  at 4 μg/ml in PBS at 4 °C overnight. 50 μl per well of B cell culture supernatants was then added. Afterwards, 50 μl per well of HRP-conjugated goat anti-Human IgG1  at 1:2,000 dilution was added as secondary antibody. Reaction was read at 450 nm on VICTOR Multilabel Counter . Purified human IgG1 whole molecule of known concentration  with 3 fold dilutions starting at 10 μg/ml was used as standard curve.50 and hybrids binding measurements, purified antibody was initially diluted at 1 μg/ml followed by serial dilution (1:2) across a polypropylene 96-well plate in PBS. The EC50 titers represent the concentration for 50% of maximal binding. We chose the OD reading of 26D1/H16.V5/1A4 at concentration of 12 ng/ml for surface loop identification in hybrid VLPs binding assay.Intact HPV16 capsids and hybrid VLPs (3 μg/ml) were coated onto 96-well microtiter plates  in 20 mM phosphate at pH 7.4 and 0.3 M NaCl, and 100 μl B cell culture supernatants were added. For EC4 cells per well for 6 h. 26D1 were subjected to a 2-fold dilution. PsVs were diluted to 2 × 105 TCID50/μl. Sixty μl of the PsV diluent and 60 μl of the serially diluted antibody were mixed and incubated at 4 °C for 1 h. The negative control was prepared by mixing 60 μl of the PsV diluent with 60 μl of the culture medium. Then, 100 μl of the above mixtures were added designated wells and incubated at 37 °C for 72 h. Flow cytometry was used to detect the number of HPV-pseudovirus-infected enhanced green fluorescent protein (EGFP)-expressing cells. The endpoint titers were calculated as the log10 of the highest dilution with a percent infection inhibition higher than 50%.HPV16 pseudo-viruses were produced according to previous studies5051Affinity measurement was conducted at 25 °C on a Biacore 3000 instrument . Mouse anti-human IgG (25 μg/ml) was directly immobilized to the surface of a CM5 sensor chip via covalent amine coupling. HPV16 VLP protein was initially diluted as 20 nM and then serial diluted to five concentrations  in 250 μl by using 0.5 M NaCl, 5 mM histidine buffer, 0.02% PS80, pH 6.2. Next, 5 μg/ml of 26D1 was loaded onto sensor chip for 60 s at a flow rate of 5 μl/min. Then each concentration of HPV16 VLP analyte was loaded onto sensor chip to carry out an association step with 26D1 ligand for 120 s for determining their relative on-rates; this was followed by a dissociation step for 600 s for determining their relative off-rates. 10 mM glycine-HCl (pH 1.7) was used as regeneration buffer at a flow rate of 10 μl/min for 30 s. The sensorgram was recorded and subjected to reference and buffer subtraction and then evaluated using BIAevaluation software.The analysis was performed with a Biacore 3000  equipped with CM5 sensor chips. The HPV16 L1 VLPs (20 μg/ml in 400 μl of PBS buffer) were covalently coupled to the CM5 sensor surface at a flow rate of 5 μl/min. Antibody saturation of the bound HPV16 L1 VLPs was achieved for 26D1 after three injections of 100 μg/ml antibody diluted in 50 μl PBS. VLP binding competition between 26D1 and H16.V5 was established by successively injecting H16.V5 three times at 100 μg/ml diluted in 50 μl PBS to the 26D1 saturated sensor surface. Similarly, we reciprocally assessed saturated binding level for 26D1 with H16.V5 pre-saturation. Then, HPV16:6 hybrid VLPs that substituted HPV6 hypervariable DE and FG loop were respectively loaded on the sensorchip for epitope competition between 26D1 and H16.V5, as described above.Eight patient sera and eight serum samples from vaccinees with different neutralization titers were randomly selected for 26D1 inhibition study. Sera blocking experiments were conducted with the saturation amount of serum after 26D1 blocking as described above. Binding was measured as a change in resonance units (RU) on the chip. Bovine serum albumin was used as a control for nonspecific binding.The initial 26D1 3D structure model was generated using the Homology Module of Discovery Studio 4.1 (Accelrys Inc.) using the CDR sequence information listed in 36Type-specific amino acids in hypervariable loops of the HPV16 and HPV6 L1 capsid proteins were identified by CLUSTAL amino acid sequence alignment 58. To ge2SO4. The optical density was measured spectrophotometrically at 450 nm.The human monoclonal antibodies 26D1 and H16.V5 were used for the heparin competitive binding ELISA. Briefly, HPV16 VLP was coated on a 96-well plate overnight at 4 °C. After HPV16 VLP coating, a total of 100 μl of 55.55 mg/ml heparin was added to the first well and a 2-fold serial dilution was used for the following wells in the column. After a 30-min incubation at room temperature, 200 ng/ml 26D1 was added to each well and incubated for 1 h. The plate was washed 5 times, and goat anti-human IgG-HRP diluted in enzyme dilution buffer was then added, followed by incubation for 1 h. After washing 5 times, the plate was incubated with a solution of tetramethylbenzidine substrate for 10 min. The reaction was stopped with 2 M HApproximately 15 μl of HPV16 L1 VLPs or hybrid VLPs at 200 μg/ml was absorbed onto carbon-coated copper grids, blotted dry, and stained with freshly filtered 2% phosphotungstic acid (pH 6.4). Grids were examined under the FEI Tecnai T12 TEM at an accelerating voltage of 120 kV and photographed at a nominal 25,000 × magnification.How to cite this article: Xia, L. et al. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5. Sci. Rep. 6, 19042; doi: 10.1038/srep19042 (2016).


Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil  O-methyltransferase (N4OMT) of Nerine bowdenii, but the underlying gene was never identified Amaryllidaceae alkaloids are a group of alkaloids with many documented biological activities. This makes them valuable potential medicines several examples are the anti-cancer compounds hemanthamine and lycorine and the anti-viral compound pancratistatin O-methylnorbelladine can be applied to galanthamine biosynthesis because 4′-O-methylnorbelladine is a universal substrate for these alkaloids Narcissus incomparabilis phenylalanine was established as a precursor that contributes the catechol portion of norbelladine. This was done using radiolabeling experiments to trace incorporation of [3-14C]phenylalanine into lycorine and degradation experiments on the resulting lycorine to determine the location of the 14C label Nerine browdenii monitoring hemanthamine incorporation trans-cinnamic acid, p-hydroxycinnamic acid and 3,4-dihydroxycinnamic acid or p-hydroxybenzaldehyde before conversion into 3,4-dihydroxybenzaldehyde 14C]tyrosine incorporation into galanthamine and degradation experiments of galanthamine 14C radiolabeling studies O-methylnorbelladine is then formed by O-methylation of norbelladine N-demethylnarwedine, which is then reduced and N-methylated to yield galanthamine  in the proposed biosynthesis of galanthamine is identified in this manner and characterized.In this study, using galanthamine biosynthesis as proof-of-concept, a novel workflow is presented to streamline the identification of biosynthetic pathway genes. A Daffodil plants were collected from an outdoor plot in St. Louis, MO, with the GPS coordinates , during peak flowering and separated into leaf, bulb and inflorescence tissues. Inflorescence is considered all tissues above the spathe. The plants were collected with the permission of the corresponding author who is the owner of the private property. No endangered species were involved in this collection.S-adenosyl methionine (AdoMet), cobalt chloride, zinc chloride and manganese chloride were obtained from Fisher Scientific. Other chemicals include acetonitrile, JT Baker; InstaPAGE, IBI Scientific; ethanol 200 proof, KOPTEC; Bradford reagent, Bio-Rad; S-adenosyl-L-homocysteine, Sigma-Aldrich; deoxynucleotide triphosphates (dNTPs), New England BioLabs, Inc. (NEB); and isopropyl β-D-1-thiogalactopyranoside (IPTG), Gold Biotechnology. The norbelladine N-methylnorbelladine, 4′-O-methyl-N-methylnorbelladine and 4′-O-methylnorbelladine were synthesized previously Formic acid, potassium phosphate monobasic, potassium phosphate dibasic, tris(hydroxymethyl)aminomethane, glycerol, sodium acetate, sodium chloride, tetramethylethylenediamine, calcium chloride, magnesium chloride and β-mercaptoethanol were obtained from Acros Organics. Glycine, papaverine hydrochloride, 2O and solvent B was 0.1% formic acid in acetonitrile. At the beginning of the program, solvent B was held at 15% for 2 min, followed by a linear gradient to 43% B at 15 min, 90% B at 15.1 min, 90% B at 20 min, 15% B at 21 min and 15% B at 26 min. A Turbo Ion Spray ionization source temperature of 500°C was used with low resolution for Q1 and Q3. All multiple reaction monitoring (MRM) scans were performed in positive ion mode. The ion fragment used for quantitation of galanthamine was 288.00 [M+H]+/213.00 [M-OH-C3H7N]+•m/z. Galanthamine was identified by comparison of retention time and fragmentation pattern to authentic galanthamine standard. The Analyst 1.5 software was used to quantitate galanthamine using a comparison of peak area of the unknown to authentic galanthamine.Daffodil leaf, bulb and inflorescence tissues were extracted by grinding tissue with a mortar and pestle cooled with liquid nitrogen. Each ground sample was split into three technical replicates. Two volumes of 70% ethanol were added followed by vortexing 5 min and centrifuging at 14,000×g for 10 min. The supernatant was filtered through a 0.2 µm low protein binding hydrophilic LCR  membrane. For galanthamine quantitation, samples were diluted 1000 fold. Liquid chromatography samples were injected (10 µl) onto an LC-20AD (Shimadzu) with a Waters Nova Pak C-18 (300×3.9 mm 4 µm) column coupled to a 4000 QTRAP (AB Sciex Instruments) for MS/MS analysis. The gradient program had a flow rate of 0.8 ml/min; solvent A was 0.1% formic acid in HThe transcriptome was generated via data cleaning, short read assembly, final assembly, and post processing steps. A modified TRIzol RNA isolation method found as protocol number 13 in Johnson et al. was used to obtain RNA for cDNA library preparation http://www.medplantrnaseq.org. Anomalies in the number of reads per contig and abnormally long or short contigs were manually curated. To normalize for read depth, each expression value for each contig was divided by the total reads for the respective tissue and multiplied by 1 million.Reads were assembled in the following manner. ABySS was used to run multiple assemblies of the reads with a range of kmers 24≤k≤54. The resulting assemblies were assembled into scaffolds using ABySS scaffolder −9 to identify methyltransferase homologs http://www.medplantrnaseq.org.Relative expression data was compared to the levels of galanthamine in daffodil tissues using HAYSTACK with a background cutoff of 1, correlation cutoff 0.8, fold cutoff 4 and p-value 0.05 Sequences found in NpN4OMT sequence were completed using Rapid Amplification of cDNA Ends (RACE) with the Invitrogen RACE kit. For gene specific primers (GSP) see The 5′ and 3′ ends of the NpN4OMT open reading frame was performed with 5.1 ng/µl daffodil bulb oligo(dT) primed cDNA, 0.4 mM dNTPs, 0.4 µM each forward and reverse outer-primer, 1 UNEB Phusion High-Fidelity DNA Polymerase and recommended buffer in a 50 µl reaction. With the following PCR program parameters: 30 s 98°C 1 cycle; 10 s 98°C, 30 s 52°C, 1 min 72°C for 30 cycles; 5 min 72°C 1 cycle. The inner-primer PCR used 1 µl of the outer-primer PCR product and used the inner-primers in Amplification of the NpN4OMT was cloned into the pET28a vector with the NotI and NdeI restriction sites that were added to the 5′ and 3′ ends of the open reading frame using the inner PCR primers. PCR product and pET28a were digested with NotI and NdeI enzymes, followed by gel purification and ligation with the T4 DNA ligase. The resulting construct was transformed into E. coli DH5α cells and screened on Luria-Bertani agar plates with 50 µg/ml kanamycin. Resulting colonies were screened by colony PCR with T7 sequencing and T7 terminator primers and Taq DNA Polymerase. The following cycle program was used: 3 min 94°C 1 cycle; 30 s 94°C, 30 s 52°C, 2 min 72°C 30 cycles; 7 min 72°C 1 cycle. Plasmid minipreps were obtained using the QIAGEN QIAprep Spin Miniprep Kit. After Sanger sequencing of constructs (Genewiz), the desired plasmids were transformed into E. coli BL21(DE3) Codon Plus RIL competent cells. The sequences of the resulting five variants have the following accession numbers KJ584561(NpN4OMT1), KJ584562(NpN4OMT2), KJ584563(NpN4OMT3), KJ584564(NpN4OMT4) and KJ584565(NpN4OMT5).E. coli and purification with TALON resin followed the protocol found in E. coli cell line containing the hexahistidine-tagged methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) construct from Choi-Rhee and Cronan’s work was used to purify Pfs protein Recombinant protein production in 1 L of NpN4OMT1 contained 10 µg of pure protein with 200 µM AdoMet, 100 µM norbelladine and 30 mM potassium phosphate buffer pH 8.0 in 100 µl. The assays were incubated for 2 hr at 30°C. The vector control was an E. coli extract purified with TALON in the same way as the methyltransferase protein. For the vector control assay, an equal volume of the pure vector control extract was substituted for the NpN4OMT1 protein in the enzyme assay. These assays were quenched by adjusting the pH to 9.5 with two volumes of sodium bicarbonate and extracted with two volumes ethyl acetate two times. After drying, the extracts were re-suspended in the initial mobile phase of the HPLC program. The HPLC separation of the assays was performed using a phenomenex Luna C8(2) 5 µm 250×4.6 mm column with solvent A (0.1% formic acid in H2O) and solvent B (acetonitrile). The program started with 10% solvent B and a flow rate of 0.8 ml/min, a linear gradient began at 2 min to 30% at 15 min, 90% at 15.1 min, 90% at 20 min, 10% at 21 min and 10% at 28 min. Injection volume was 20 µl using a Waters Autosampler. Waters UV detector was set to 277 nm.Enzyme assays for initial testing of 2 added and the temperature was increased to 37°C in 100 µl total reaction volume. When performing kinetic assays, the E. coli enzyme Pfs was added to break down SAH and prevent product inhibition. Product formation in kinetic experiments was quantified by comparing product peak area to a standard curve of the expected product or equivalent. Papaverine was used as an internal standard.After optimization of the assay, the buffer was changed to 100 µM glycine at pH 8.8, with 5 mM of MgCl+/138.00 [M-C8H9O]+•m/z, 260.00 [M+H]+/121.00 [M-C7H8NO2]+•m/z, 274.00 [M+H]+/137.00 [M+H-C8H9O2]+m/z, 274.00 [M+H]+/122.00 [M+H-C8H10NO2]+m/z. The fragments with 260.00 [M+H]+m/z and 274.00 [M+H]+m/z molecular ions were replaced when looking at N-methylnorbelladine for 274.00 [M+H]+/152.10 [M-C8H9O]+•m/z, 274.00 [M+H]+/121.00 [M-C9H12NO2]+•m/z, 288.00 [M+H]+/150.10 [M-C8H9O2]+•m/z and 288.20 [M+H]+/137.00 [M-C9H12NO]+•m/z. Papaverine internal standard was monitored with the following fragments 340.40[M+H]+/324.20 [M-CH3]+•m/z and 340.40 [M+H]+/202.10 [M-C8H9O2]+•m/z. When conducting dopamine kinetics, galanthamine was used as the internal standard and samples were not ethyl acetate extracted prior to LC/MS/MS analysis. To remove protein, two volumes of acetonitrile were added followed by 1 hr at −20°C and 10 min centrifugation at 16,100×g, 4°C. The supernatant was dried under vacuum and re-suspended in the starting mobile phase before analysis. The HPLC time program was changed to start at 5% solvent B with solution going to waste until 3.9 min, at 5 min start linear gradient to 25% B at 25 min, 90% B at 9.5 min, 90% B at 11 min, 5% B at 11.1 min and 5% B at 16 min. Ions monitored in the MRM were 168.00 [M+H]+/151.00 [M+H-OH]+m/z and 168.00 [M+H]+/119.00 [M-OH-OCH3]+•m/z. AdoMet steady state kinetic parameters were determined with norbelladine as the saturated substrate. Product was quantitated using HPLC with the 28 min program used for screening enzyme assays. Product for assays on the additional NpN4OMT variants was detected with this same 28 min program on HPLC.With the same solvent system as for screening enzyme assays, the HPLC program started with 20% B and a flow rate of 0.8 ml/min, a linear gradient began at 2 min to 25.4% B at 7 min, 90% at 7.2 min, 90% at 9 min, 20% at 9.1 min and 20% at 14 min. A 4000 QTRAP mass spectrometer coupled to the same LC column and time program as used in HPLC was used to collect all compound mass and fragmentation data. For fragmentation data and program setting details see mK was at least five fold higher than the minimum concentration of substrate and fivefold lower than the maximum concentration of substrate tested. mK and catk were calculated by nonlinear regression to the Michaelis-Menten kinetics equation with the GraphPad PRISM 5.0 software.When conducting kinetic experiments the O-methylnorbelladine in CD3OD at 600 MHz on a BrukerAvance 600 MHz spectrometer equipped with a BrukerBioSpin TCI 1.7 mm MicroCryoProbe. Proton, gCOSY, ROESY, gHSQC, and gHMBC spectra were acquired; 13C chemical shifts were obtained from the HSQC and HMBC spectra. Chemical shifts are reported with respect to the residual non-deuterated MeOD signal (δH 3.31) (NMR spectra were acquired for 4′-δH 3.31)  and S5.T values relative to standard ribosomal RNA and leaf tissue cDNA for leaf, bulb and inflorescence tissues of daffodil were created using 1 µg RNA from the respective tissues, random primers and M-MLV reverse transcriptase according to the Invitrogen protocol. qRT-PCR was conducted with a TaqMan designed gene expression assay for the methyltransferase with ribosomal RNA as a reference according to manufacture protocol. Reactions (5 µl) were performed in quadruplicate with outlier exclusion using Applied Biosystems StepOnePlus Real-Time PCR system. Methyltransferase relative expression values were determined by calculating ΔΔCChlorophytum borivilianumThe Illumina sequencing of daffodil leaf, bulb and inflorescence tissues resulted in 65 million paired-end reads that were used to make the daffodil transcriptome assembly. The transcriptome assembly consisted of 106,450 sequences  with a mThe LC/MS/MS data for leaf, bulb, and inflorescence tissues resulted in a pronounced accumulation pattern of galanthamine. The largest concentration was found in bulb tissue, with a lower level found in leaf and the lowest level in inflorescence .NpN4OMT that fits the HAYSTACK model (NpN4OMT was found to be a 239 amino acid protein with a predicted molecular weight (MW) of 27 kDa. When expressed using the pET28a vector, the added N-terminal Histidine tag increased the MW to 29 kDa  and H-6′ (δH 6.90) to the same carbon (δC 149.9) placed the methoxyl group at C-4′. Its location was further supported by a ROESY correlation from the methoxyl protons to H-5′ (δH 6.98). The NMR data thus confirmed that 4′-O-methylnorbelladine is the product of the enzyme reaction  NpN4OMT than SynOMT. It is probable that the 4-OMT activity of NpN4OMT was acquired independently of SynOMT Click here for additional data file.Figure S2NpN4OMT1 product 4′-O-methylnorbelladine COSY spectra.(TIF)Click here for additional data file.Figure S3NpN4OMT1 product 4′-O-methylnorbelladine HMBC spectra.(TIF)Click here for additional data file.Figure S4NpN4OMT1 product 4′-O-methylnorbelladine ROESY spectra.(TIF)Click here for additional data file.Figure S5NpN4OMT1 product 4′-O-methylnorbelladine HSQC spectra.(TIF)Click here for additional data file.Figure S6Protein sequence alignment of NpN4OMT variants. Five unique variants of the NpN4OMT sequence are aligned against the original sequence predicted by the de novo assembled transcriptome using CLC software. Dots are identical residues.(TIF)Click here for additional data file.Figure S7Effect of divalent cations, temperature and pH on NpN4OMT1 enzyme activity. (A) Divalent cations tested with 5 min assays with 5 µM of cation Ca2+, Co2+, Zn2+, Mg2+ or Mn2+. (B) pH optimum 15 min assays with 5 µM Mg2+. (C) Temperature optimum 15 min assays with 5 µM Mg2+. Divalent cation and pH testing reactions are 100 µl reactions at 37°C. The divalent cation test contained 4 µM norbelladine, while pH and temperature optimum tests contained 100 µM norbelladine in the assay mix.(TIF)Click here for additional data file.Table S1Methyltransferases used in BLAST search.(DOCX)Click here for additional data file.Table S2Methyltransferases used in phylogeny.(DOCX)Click here for additional data file.Table S3Primers used in RACE, cloning and colony PCR.(DOCX)Click here for additional data file.Table S4Parameters used for LC/MS/MS analysis.(DOCX)Click here for additional data file.


Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11–13%, i.e., 34–41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S. Nevertheless, action appeared required to carefully estimate the H2S concentrations derived from the H2S donor  used in our experiments.The physiological concentrations of H2+ as well as by membrane voltage and are modulated by a wide variety of intra- and extracellular factors, including protein kinases, phosphatases causing phosphorylation/dephosphorylation or changes in their redox environment in particular at the C-terminal region . We used these three different but related cell lines, because they all express BK channels who differ in their sequences and respond differently to phosphorylation or in the responsiveness to arachidonic acid. The sequences of BK α-subunits in GH3 and GH4 splice variants differ most prominently by the presence or absence of 27 amino acids in the COOH terminus of the channel  and prolactin and express various receptors. Stressors applied to GH cells lead to the expression of a 59 amino acid cysteine rich insert at the pore forming α-subunit C-terminus of BK channels—termed GH STREX cells  and hence destabilization of the reference electrode. Recordings from excised outside-out patches were made with an Axopatch-200B amplifier connected to a Digidata 1322A interface using pClamp10 software for data acquisition and analysis. Data were filtered at 1 kHz offline and analyzed with Clampfit software . Raw recordings were idealized with the built in feature of pClamp's Clampfit module using the half-amplitude threshold method with automatic baseline and level tracking. Dwell time analysis was done by fitting open dwell time distributions to standard exponentials with the appropriate number of terms to get an optimal fit. Since BK channels are localized in clusters in cell membranes, all of our patches contained more than one channel. For this reason we analyzed only open dwell times but not closed channel kinetics. Single channel current amplitudes were calculated by fitting amplitude histograms to a Gaussian distribution. Channel open probability was expressed as Popen = NPo/n, where NPo = [(to)/(to + tc)], Po = open probability for one channel, to = sum of open times, tc = sum of closed times, N = actual number of channels in the patch, and n = maximum number of individual channels observed in the patch. All equations used were standard built in equations from Clampfit. Experiments were repeated at least three times and the mean, as well as the s.e.m. (standard error of the mean) were calculated.In brief: pipettes for single channel patch clamp recordings were drawn from borosilicate glass (Science Products/FRG) with resistances of 3–6 MegaOhm . For rapid solution exchange (about 300–500 ms) membrane patches were held in a stream of the experimental solution from a second pipette.2, 1 CaCl2, 10 HEPES, pH 7.2. The regular pipette solution contained in mM: 145 KCl, 1 MgCl2, 10 HEPES, pH 7.2, and 5 EGTA, 3.63 CaCl2 – resulting in 0.5 μM free Ca2+ as calculated using the Webmaxc extended calculator (http://www.stanford.edu/~cpatton/webmaxcE.htm). Experiments were carried out at room temperature between 20 and 22°C. Sodium hydrosulfide  was used as a source of H2S. NaHS at concentrations usually used in the present study did not change the pH of the buffered solution. NaHS solutions prepared shortly before experiments were clear and were usually used 3–5 min but no longer than 20 min. For details of making and working with H2S, see Hughes et al.  0.1 units/ml; PKC inhibitor fragment 19–31 (PKCin) – 500 nM; PKG – 400 units/ml plus 50 μM cGMP; Staurosporine (STS) – 1 μM; Okadaic Acid (OA) – 100 nM.+ and HS−, and HS− associates with H+ to produce H2S. Previously as a rule of thumb we calculated for neutral solutions that one-third of NaHS exists as H2S and the remaining two-thirds are present as HS−  together with an Apollo 1000 free radical Analyser . The response time of the sensor is less than 5 s, with a sensitivity of 2 pA/nM and a detection range from 5 nM to 100 μM H2S. For calibration of the sensor instructions according to the WPI manual were used. After we had established a calibration curve we determined the rate of loss of H2S versus time for the NaHS donor. As depicted from Figure 2S obtained after 20 min is 42%. A loss of about 33% after 15 min and 90% after 30 min was reported previously by Kimura et al. . For NaHS = 300 μM and pK1 = 6.89, the H2S concentration calculates to 97 μM, which is quite similar to the rule of thumb of 1/3 H2S in solution (see above). Calculated from pK1 = 7.04 for standard conditions  K1 = 10−7.04  + (−0.157 × (S0.5)) + 0.0135 × S  = is the natural logarithm of T, and S = salinity). Taken salinity as zero for water and temperature 20°C equal to 293.15 Kelvin we calculate pK1 = 7.056, which is very close to literature value . The relationship is linear up to about 50 μM H2S, respectively 226 μM NaHS. Table 2S concentrations. During dilution from the stock to the final solution which took about 30–60 s we may have lost about 5–10% H2S by evaporation  containing the experimental fluid. The syringe opening was covered with parafilm with a small whole inserted to allow for pressure exchange. The tip of the perfusion pipette was located in the recording chamber at about 1 mm distance to the measured cell or patch. The constant perfusion rate of 1 ml/80 s ensured that the channels recorded were always submerged by the desired HThere are some problematic features of proportional data, like open probabilities (Po), which often hinder the use of parametric statistical procedures, such as ANOVAs and regression analysis. Basic assumptions of these methods are often violated, because all values coming from single channel recordings are set within the interval 0 < Po < 1. To be more precise, proportional data tend to be non-normally distributed and heteroscedastic in nature, which actually means that the size of sample variances is not evenly distributed along the interval of 0 < Po < 1.p = ln(p/(1 − p)), which “normalized” this kind of data and substantially reduced heteroscedasticity. After this transformation, “usual” parametric methods could be used. Due to similar reasons, dwell-time measurements were also log-transformed prior to statistical analysis.Besides using less powerful non-parametric methods, there are other strategies to overcome these limitations, e.g., data transformation , high significant , and most significant . Data described in the text as percentage were calculated by setting control values to 100% and expressing experimental values as percentual change of controls.Evaluation of statistic analysis is given as: significant  (Table ***) different to ATP0. PKC  in the pipette solution decreased BK Po non-significantly compared to ATP1, but Po was significantly different from ATP0 (*). PKC is known to cause inhibition of BK channel activity from GH cells  if compared to ATP1. The experiments support previous results of an inhibitory role of PKC at BK channels from GH3 cells  for GH3, GH4, and GH4 STREX cells are listed in Table *) Table . Similar*) Table , and rev*) Table . All fur*) Table , however***) (Table **) with high significance compared to ATP1, as previously reported by Zhou et al. (All drug concentrations used for these cells were similar to those used with GH3 cells. With ATP1 Po increased high significantly compared to ATP0 (*) Table . Using Pu et al. . Use of u et al. . With th+ conductance (RCK1 and RCK2). This insert bears various cysteine residues which can be phosphorylated. The STREX insert speeds activation and slows deactivation kinetics and their half-activation voltage is shifted to the left . The increase caused by ATP was, however, less compared to BK channels from GH3 or GH4 cells (Table ***) when compared to all other conditions, implying that inhibition of the BK channel attached phosphatase allows for unrestricted phosphorylation of the channel protein.These cells contain a 59 amino-acid insert, so called STREX (stress axis regulated) exon at the BK channel α-subunit C terminus, located between the two regulatory domains of Kls Table . PKA is ls Table . PKA inc2S effects are shown in Figure We again present data separately for all three cell types used in our study. Original recordings of H2S concentration 34–41 μM, see Materials and Methods), applied to the extracellular bath solution most significantly increased Po of BK channels from 0.01522 ± 0.00302 to 0.0295 ± 0.00722  , V = +30 mV), equal to 190.58 ± 13.81% of control (=100%) during the first minute of H2S perfusion. Po decayed after 1 min H2S application during the following 3 min. A similar transient response was observed previously using ethanol  with NaHS, i.e., to 191.65 ± 23.52% of control (=100%) , i.e., to 169.42 ± 19.99% of control. PKG (50 units/ml) also increased Po from 0.0724 ± 0.0118 (control) to 0.1395 ± 0.0236(n = 8) in NaHS by 205.56 ± 19.08% (***). PKC catalytic subunit  decreased BK channel activity to 0.04434 ± 0.00752 and NaHS had no significant effect . After addition of PKC inhibitor pseudo-substrate , Po was 0.07408 ± 0.01808 (control), but NaHS again had no significant effect . During application of the kinase inhibitor staurosporine  Po was decreased to 0.04683 ± 0.01493, and in this case NaHS significantly increased Po to 0.07483 ± 0.01892 , i.e., to 168.56 ± 11.87% of control. The most significant increase of Po compared to either ATP0 or ATP1 was obtained with the phosphatase inhibitor okadaic acid (100 nM) which increased Po to 0.3075 ± 0.0581, however, Po was not further significantly altered by NaHS .As reported previously  Figure . All furn = 8 (***), i.e., to 183.48 ± 22.12% (***) after application of NaHS. With ATP1 solution, Po increased significant from 0.11647 ± 0.01969 (control) to 0.20591 ± 0.02146, in NaHS, n = 11 (***), i.e., to 201.36 ± 19.12%. After application of PKA, Po was 0.12569 ± 0.02224 (control), but with NaHS no significant alteration of Po 0.12693 ± 0.02414, n = 11, i.e., to 107.33 ± 11.86% was observed. With PKG Po increased highly significant from 0.05229 ± 0.01732 (control) to 0.1430 ± 0.04916 (n = 7) in NaHS, i.e., by 258.95 ± 33.78% (***). Application of PKC subunit  decreased Po to 0.02771 ± 0.00647 and NaHS increased Po significantly to 0.0465 ± 0.01669, i.e., to 161 ± 28%, n = 7 (*). A similar Po of 0.02089 ± 0.00553 was obtained with PKC inhibitor , and Po was high significantly increased with NaHS to 0.04093 ± 0.0108, n = 7 (***), i.e., to 186.78 ± 19.85%. With staurosporine  Po was 0.0505 ± 0.01474 and increased with NaHS highly significant to 0.087 ± 0.02195, n = 5 (***), i.e., to 181.66 ± 15.35%. Okadaic acid (100 nM) substantially increased Po to 0.44223 ± 0.0403 compared to either ATP0 or ATP1 controls, whereas NaHS decreased Po under this condition to 0.31953 ± 0.03142, n = 7 (***), i.e., to 74.841 ± 7.8%.All basic conditions and concentrations of drugs were similar to the experiments using GH3 cells. In ATP0, Po increased highly significant from 0.00968 ± 0.00285 (control) to 0.01632 ± 0.00452, n = 7, i.e., to 148 ± 11.99%. With ATP1 Po increased to 0.03428 ± 0.00931, n = 13 compared to ATP0, but this increase was much less compared to Po-values obtained in GH3 and GH4 cells , i.e., to 177.74 ± 15.91%. PKA subunit (50 units/ml) increased Po to 0.08221 ± 0.03214, n = 8 under control conditions, however, NaHS application had no significant effect on Po, 0.05739 ± 0.02129, n = 8, i.e., 78.65 ± 9.38% of control. PKG high-significantly increased Po from 0.05043 ± 0.01330 (control) to 0.08829 ± 0.01635 in NaHS, by 201.94 ± 40.37%, n = 8 (***).In ATP0 BK channel Po of 0.00652 ± 0.00224 (control) was low compared to Po of GH3 and GH4 cells. NaHS non-significantly increased Po to 0.00921 ± 0.00286, n = 6 (control), however, NaHS increased Po to 0.06833 ± 0.01267, i.e., to 322.44 ± 74.48%, although with low significance (*). With PKC inhibitor  Po of 0.0400 ± 0.00879, n = 5 was similar to ATP1, and increased with NaHS to 0.10552 ± 0.02854, n = 5, i.e., to 253.07 ± 28.82%, however not significantly. Also staurosporine  non-significantly increased Po from 0.0375 ± 0.00975 (control) to 0.10114 ± 0.02397 in NaHS, n = 8, i.e., to 306.14 ± 49.76%. In okadaic acid (100 nM) which significantly increased Po to 0.39927 ± 0.09621 compared to ATP1, NaHS had no additional significant effect on Po, 0.42796 ± 0.08584, n = 7, i.e., to 114.93 ± 9.22%.As with previous results from GH3 and GH4 cells, using PKC subunit  BK channel Po was low, 0.02533 ± 0.00604, 2S, whereas BK channels from GH4-STREX cell did not respond to H2S. Po of BK channels of all cell types phosphorylated by ATP was highly significantly increased and H2S further increased Po at high significance. PKA and PKG were not able to increase Po beyond the ATP1 effect. H2S was able to increase Po of BK channels from GH3, but not those of GH4 and GH4 STREX cells. H2S high significantly increased Po of all cell types pretreated with PKG. PKC-subunit (PKCsu) either had no effect (GH4-STREX) or decreased Po  but H2S increased Po of BK channels pretreated with PKCsu in GH4 and GH4 STREX but not those of GH3 cells. Staurosporin had no effect on BK channel Po activity but did not prevent the activating effect of H2S. Okadaic acid most significantly increased BK channel Po of all cell types and H2S had no further effect on channel Po, it even decreased BK channel Po of GH4 cells.In summary, our result show Table  that Po ***) increased mean open dwell times from 0.74 ± 0.087 ms (ATP0) to 2.022 ± 0.18 ms (ATP1). Open dwell times were also significantly increased with PKA to 1.78 ± 0.22 ms (***), with PKG to 1.56 ± 0.15 ms (**) and with PKC subunit to 1.78 ± 0.6 ms (*), compared to an ATP0 solution. A significant increase of mean dwell times was also observed with BK channels from GH4 cells from 0.77 ± 0.10 ms (ATP0) to 1.49 ± 0.11 ms (**) after application of ATP1. With PKA dwell times increased to 3.00 ± 0.44 ms (***) and with PKG to 1.54 ± 0.12 ms (**) if compared to ATP1 , PKCin vs. PKA (***), staurosporine vs. PKA (***) and okadaic acid vs. PKA (***) and PKG vs. PKA (*). NaHS had a significant increasing effect on open dwell times only in okadaic acid (***). In GH4 STREX cells open dwell times were only significantly increased when PKG is compared with ATP0 (*).Values of BK channel Po, amplitude and dwell time are shown in Figures 1 Figure . Signifi**), with GH4 cells in okadaic acid from 0.10 ± 26 ms to 1.73 ± 0.46 ms (***), and in ATP0 in GH4 STREX cells from 0.69 ± 0.10 ms to 0.99 ± 0.16 ms (*). NaHS had no effect at dwell times with all other experimental settings  to (1) obtain reliable, well visible effects at the channels, and (2) being able to compare our results with previous investigations. Previous studies  increased Po was about 30 μM NaHS, which effectively amounts to 3.4–4.1 μM free H2S. Hence, H2S appears to induce BK channel activation at a physiological relevant concentration range. The effective H2S level at its target site(s) is unknown, however. H2S may be stored as bound sulfane sulfur and/or neuronal excitation can trigger its release from surrounding astrocytes  applied for a few minutes were completely reversible  at BK channel Po  was only about 6–8%. As indicated from a previous dose-response plot , HS− (~80%), and traces of S2−, are always simultaneously present in the solution or in the cytosol. This mixture is usually referred to in the literature as H2S for simplicity  which in turn may prime the channels for H2S activation.With pipette solutions containing no ATP H2S only had a high significant effect on BK channel Po from GH3 cells but had no effect on GH4 and GH4 STREX cells. Phosphorylation of BK channels by PKA therefore appears to prevent further H2S activation in these cells. PKG had no significant effect on Po compared to ATP1 of all cell types , whereas NaHS significantly increased Po of BK channels phosphorylated by PKG of all cell types. In all of our experiments the initial phosphorylation state of BK channels at the start of experiments was not determined and remains unknown. Also the amount of phosphorylation by ATP or the addition of kinases was not measured. This should be kept in mind when considering the interpretation of the results.In all three cell lines PKA effect on Po is not significant different compared to ATP1. The result indicates that the channels are already sufficiently phosphorylated in the presence of ATP. PKA has been reported to activate, inhibit or have no effect on BK channels as reviewed by Schubert and Nelson . These d2S in this context did not alter BK channel Po from GH3 cells, but increased Po of GH4 and even more of GH4 STREX cells. With PKC inhibitor, H2S had no effect at GH3 cells but high significantly increased Po of BK channels from GH4 and GH4 STREX cells. Although PKC phosphorylation suppresses Po of BK channels H2S appears able to overcome this effect and to stimulate the channels. The kinase inhibitor, staurosporine, had no significant effect on BK channel Po of all three cell lines. H2S under these conditions was able, however, to significantly increase Po. Hence, in all three cell types Po of non-phosphorylated (ATP0) and phosphorylated channels by ATP1 and by PKG can be elevated by H2S. PKC phosphorylation prevented the Po increasing H2S effect in GH3 cells but increased Po in GH4 and GH4 STREX cells. PKA also differently affected cell types with increasing Po of GH3 cells but not of GH4 and GH4 STREX cells (which appear even decreased by H2S).PKC subunit and PKC inhibitor decrease channel Po but this effect was only significant with GH4 cells. Our data are in congruence with a decrease of channel activity by PKC reported previously . This peculiar action of okadaic acid may be due to the fact that a very active protein phosphatase is associated with the BK channels  channel Po is increased. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX but increased BK Po of GH3 cells at high significance. This suggests that PKA phosphorylates the channels of GH4 and GH4 STREX cells at some other site which rearranges the protein in a way that H2S is no longer able to act at the channels. Zhou et al.  or if phosphorylated by ATP can be activated by Hu et al.  showed t2S on BK channels Po which may be dependent on the channels conformation which exposes or impedes phosphorylation sites and this way may govern the access of H2S to reach its effective location.Our experimental results indicate that phosphorylation may prime or prevent the action of HThe 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.


The effects of temperature on the structure and performance of the as-prepared cathode material are systemically studied by X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (TEM), galvanostatical charge/discharge and electrochemical impedance spectra. The results show that a spinel Li4Mn5O12 component can be controllably introduced into the lithium-rich layered material at 750°C. Besides, it has been found that the obtained layered-spinel cathode material represents excellent electrochemical characteristics. For example, it can deliver a high initial discharge capacity of 289.6 mAh g−1 between 2.0 V and 4.6 V at a rate of 0.1 C at room temperature, and a discharge capacity of 144.9 mAh g−1 at 5 C and 122.8 mAh g−1 even at 10 C. In addition, the retention of the capacity is still as high as 88% after 200 cycles, while only 79.9% for the single-phase layered material. The excellent electrochemical performance of the as-prepared cathode material can probably be attributed to the hybrid structures combining a fast Li-ion diffusion rate of 3D spinel Li4Mn5O12 phase and a high capacity of the layered Li-Mn-Ni-Co-O component.Homogeneous lithium-rich layered-spinel 0.5Li The huge irreversible capacity loss has been attributed to the elimination of oxygen atom and lithium ion vacancies from the layered lattice at the end of the first charge4+ ions and the thick SEI layer formed by a reaction of the cathode surface with the electrolyte5Lithium-rich cathode materials xLi2MnO3·−xLiMO2  compared with that of the layered material (71%). Besides, WuxMn2−xO4 or Li1+xMnO4 membrane for high-energy and high-power lithium-ion batteries. Obviously, lithium-rich cathode materials with layered-spinel hybrid structures are currently becoming the research hotspots. However, it is worth thinking how to integrate the controlled spinel components into the layered materials and what kind of spinel materials will maximally improve the comprehensive performance of composite materials.Currently, spinel lithium-ion battery cathodes with three-dimensional (3D) interstitial space for diffusion of lithium ions have also attracted more and more attention due to its high discharge potential and excellent rate capability74Mn5O12 component via a solvothermal process and subsequent simple heat treatment. As the the Li4Mn5O12 spinel has been prepared at a low temperature154Mn5O12 phase to Li2MnO3 phase when the temperature was raised above 700°C4Mn5O12, which has a three-dimensional (3D) interstitial space permiting fast diffusion of lithium ions and stable tetravalent manganese+ insertion/extraction framework of the spinel component. However, we have found that during the calcinations process the temperature of solid phase reaction will dramatically influence the performance of the material. Thus, in this paper, the effects of preparation temperature on the structure and performance of the as-prepared cathode materials were systemically studied.Recently, our group has designed and synthesized lithium-rich layered materials with improved electrochemical performances12132CO3, the resultant cathode materials still maintained a good spherical morphology like precursor particles. In addition, each of the spherical cathode particles was made up of a large number of primary grains. A close observation shows that the primary grains of the samples are strongly influenced by the calcinations temperatures. It can be seen from The typical surfaces and morphologies of the carbonate precursor and corresponding cathode materials prepared under the different calcination temperatures are examined by SEM, as shown in C2/m) and spinel hybrid structure. The spinel peaks can be well indexed to cubic Li4Mn5O12 with space group of Fd3m (PDF#46-0810) marked as plum blossom pattern in 2MnO3 (C2/m) between 20° and 25° become more clear accompanied with the increasing amounts of the layered phase (from D750 to D800). When the calcinations temperature increases, the D900 and D850 samples show a typical α-NaFeO2 layered structure (space group 2MnO3 character (space group C2/m) with some weak peaks between 20° and 25°. Particularly, the clear splitting of the (006)/(012) and (018)/(110) peaks indicates that the two samples have a well-organized layered structure4Mn5O12 (Fd3m) and layered phases (2MnO3 (C2/m) between 20° and 25° disappear for the D700 sample. The above results demonstrate that the annealing of Li4Mn5O12 at the temperature above 700°C leads to existence of monoclinic Li2MnO3, which is in consistence with the previous reports16172 layered structure with monoclinic Li2MnO3 structure (C2/m).The X-ray diffraction (XRD) patterns of as-prepared spherical cathode materials calcined at various temperatures are shown in 12) and 0/(110) pe4Mn5O12 structure for the (Fd3m) space group taken from along (400), (511) and (111) directions. From the d-spacing of 2.03 A corresponded to (104) plane of α-NaFeO2 layered structure and 2.04 A corresponded to (400) plane of (Fd3m) Li4Mn5O12 structure. In addition, the D750 sample have spinel and layered crystalline with the characteristic plane (400) and (111) of (Fd3m) Li4Mn5O12, the (020), (022) plane of (C2/m) Li2MnO3 and the (104) plane of α-NaFeO2 layered structure . The HRTEM images and corresponding fast Fourier transformation (FFT) of the D700 sample is shown in −1 current density). The D750, D800, D850 and D900 samples show a representative profile of lithium-rich layered materials with a sloping curve below 4.5 V and a long plateau around 4.5 V in the first charge process. The sloping curve below 4.5 V corresponds to lithium ions extraction from the layered LiMO2 component with the concomitant oxidation of Ni2+/Ni4+ and Co3+/Co4+, while the long plateau ~4.5 V is consistent with the removal of lithium ions and oxygen from the Li2MnO3 component234Mn5O12 had been decomposed into monoclinic Li2MnO3 when calcined at 700°C−1, whereas the D700, D800, D850 and D900 cells exhibit an initial discharge capacity of 221.2, 270.104, 247.54 and 230.757 mAh g−1, respectively.To compare the electrochemical performance of the as-prepared materials, coin cell tests are performed using the D700, D750, D800, D850 and D900 samples as the cathode materials and Li metal as a counter electrode. −1 at 0.5 C, 144.9 mAh g−1 at 5 C and 122.8 mAh g−1 even at 10 C. However, the discharge capacity of D700 material at 0.5 C, 5 C and 10 C are only 180.2 mAh g−1, 60.7 mAh g−1 and 33.2 mAh g−1, respectively. Besides, the D800, D850 and D900 have a better rate performance than D700 but doesn't appear as much improvement as D750 materials. Although the D750 and D800 both have the lithium-rich layered and spinel Li4Mn5O12 phases, the rate performance is improved more obviously when the calcinations temperature is controlled at 750°C.The rate capabilities of these cathode materials are shown in −1 at 0.5 C after 200 cycles. Meanwhile, the discharge capacity of D750 reaches 223.6 mAh g−1 at 0.5 C after 200 cycles between 2.0 and 4.6 V with a capacity retention of 88%, which has been significantly improved with the introduction of spinel structure−1, and the capacity retentions is 86.5% after 200 cycles. Whereas, the discharge capacities of D850 and D900 are just 172.1 and 159.7 mAh g−1 with the capacity retentions 79.9% and 77.9%,respectively. The cycling performance of the five samples is shown in Rsf) due to lithium ion diffusion in the surface layer (including SEI layer and surface modification layer)Rct)4+/Mn3+ redox couple, while the cathode peaks at about 2.6 V match along with Mn4+/Mn3+ redox. The reaction at ~2.8 V is associated with the transformation of cubic to tetragonal phase during the extraction of lithium ions from empty 16c octahedral site of the cubic spinel structure2O and another peak at ~4.0 V which is roughly correspond to the expected value for Ni2+/Ni4+ reaction. The peak at ~3.3 V indicates that the lithium ion will insert into the MnO2 which is derived from Li2MnO3 at 3.3 V (Mn4+ to Mn3+)To explain the different electrochemical properties of the samples prepared at different temperatures, electrochemical impedance spectroscopy (EIS) is carried out after being charged to 4.6 V. The corresponding Nyquist plots are given in LiD) with greater accuracy for compounds with varying composition or voltage, which has been extensively used to calculate the value of LiD in electrode materials31Vm is the molar volume of Li1.5Ni0.2Co0.2Mn0.6O2, which is 20.42 cm3mol−1 deduced from the crystallographic data. S is the active surface area of the electrode, and L is the thickness of the electrodeGITT is considered to be a reliable method to determine the diffusion coefficient of lithium ions (−1) for an interval τ of 10 min followed by an open circuit stand for 1 h to allow the cell voltage to relax to its steady-state value, Es. The procedure was repeated for the voltage window of operation 2.0–4.6 V. It can be seen that the cell voltage stabilizes to a stable value after the 1 h open-circuit stand after each current flux. At the same time, it can be found that the discharging time of D750 is much longer than the others, which indicates that the voltage drops more slowly during the discharge process and this is accord with the result of charge-discharge test. From E/dx about the two samples following the x is obvious, in the process of de-insertion the lithium, along with the increasing of x the value of dE/dx gradually rise, but the dE/dx of D800 reaches a maximum and then drops with x = 0.63, this phenomenon occurs more early than the samples of D750, this is a strong evidence that the samples can release more capacity during the discharge. DLi values of D750 and D800 samples, it is found that the DLi values of D750 and D800 are from 4.0 × 10−11 to 8.0 × 10−11 cm2 s−1 and from 2.0 × 10−11 to 4.0 × 10−11 cm2 s−1, respectively. It is obvious that the the DLi values of D750 is slightly higher than that of D800 sample, therefore the improvement of the lithium-ion diffusion coefficient can also be an explanation of the greatly improved electrochemistry performance.0.2Mn0.54Ni0.13Co0.13]O2–spinel LiMn1.5Ti0.5O4 composite cathode material, but the particle-size of the samples was not uniform and the initial discharge capacity is just 220 mAh g−1, which is apparently much lower than the herein 289.6 mAh g−1. In addition, a spinel-layered Li-rich Li-Mn-Co-O material synthesized by Li' GroupFinally, it should be also pointed out that despite of many studies on this cathode material have been conducting so far, further intensive studies are still necessary. In this paper, the uniform microspheres with layered-spinel composite structure have been synthesized and show an excellent electrochemical performance. Wang' Group−1 between 2.0 V and 4.6 V at a rate of 0.1 C, and the discharge capacity at 0.5 C, 5 C and 10 C reached 264.8 mAh g−1, 144.9 mAh g−1 and 122.8 mAh g−1, respectively. Additionally, the capacity retention is still 88% after 200 cycles when cycled at 0.5 C. The DLi (GITT) values of the D750 sample are stable at a value of ~6 × 10−11 cm2/s in the voltage range of 2.0–4.6 V. The charge transfer resistances are obviously reduced with the emergence of spinel structure. Therefore, the lithium-rich layered-spinel composite cathode material prepared at 750°C will possess enormous application prospect in the lithium ion batteries.In summary, lithium-rich layered-spinel composite cathode materials were synthesized via a solvothermal process followed by calcinations at 750°C. The calcination temperature will critically influence the crystal structure of the obtained cathode material. When the calcination temperature is equal to 700°C, the obtained material shows only a spinel structure; while the calcination temperature is above 850°C, the as-prepared material shows just a lithium-rich layered structure. Besides, the introduction of the spinel structure into the layered lithium-rich cathode materials can effectively improve the electrochemical performance of lithium-rich layered cathode material. Both of the materials obtained at 800 and 750°C show the two phases with different ratios, and we further compared the electrochemical performance of the two samples. The hybrid structure cathode material obtained at 750°C can deliver an initial discharge capacity of 289.6 mAh g2·4H2O was dissolved in 50 ml ethanediol. Then, 0.498 m mol CoCl2·6H2O and 0.498 m mol Ni(NO3 )2·6H2O was added to the solution which was stirred vigorously for 0.5 h in the Teflon wares. At the same time, the prepared NH4HCO3 solution was dropwise added into the mixed solution. Next, the sealed Teflon ware was put into a stainless steel autoclave, which was hold at 200°C for 20 h, followed by cooling at the room temperature to obtain purple precipitates. The precipitates were centrifugalized and washed by ethanol several times, the residue was dried at 60°C. Finally, the as-produced carbonate precursor was preheated at 500°C for 6 h, and mixed with Li2CO3, then calcined at 700°C, 750°C, 800°C, 850°C, 900°C for 10 h in air respectively, to form the spherical Li-rich cathode materials. To distinguish the products preferably, five kinds of compounds have been named as D700, D750, D800, D850, and D900, which correspond to reaction temperatures of 700°C, 750°C, 800°C, 850°C, and 900°C, respectively.Firstly, 2.0 m mol MnCl−1 in the 2θ range of 10°–80°. The morphologies were investigated by a JEOL JSM-6610LV scanning electron microscope. Transmission electron microscope JEOL JEM-2100F with accelerating voltage of 200 kV was used in order to obtain TEM images.The structures of samples were characterized by X-ray diffraction on a Rigaku D/MAX-2500 powder diffractometer at 40 kV and 100 mA using a graphite monochromatic and Cu-Ka radiation (k = 0.15418 nm) operated with a scan rate of 2° min6 in 1:1 ethylene carbonate (EC)/dimethyl carbonate (DMC) electrolyte. The assembly of the cells was carried out in an argon-filled glove box, where water and oxygen concentration were kept less than 5 ppm. All the cells were allowed to age for 12 h before testing. Charge-discharge measurements were performed at room temperature under different rates in a voltage range of 2.0–4.6 V on the Neware battery test system.The cathodes for testing cells were fabricated by mixing the cathode materials, carbon black, and polyvinylidene fluoride (PVDF) binder with a weight ratio of 80:10:10 in N-methyl pyrrolidinone, which were then pasted on aluminum foil followed by drying under vacuum at 110°C for 24 h. The testing cells were assembled with the cathodes thus fabricated, metallic lithium anode, Celgard 2300 film separator, and 1 M LiPFThe galvanostatic intermittent titration technique (GITT) was performed at room temperature under a small current flux of 0.1 C and a long time interval of 60 min in a voltage range of 2.0–4.6 V on the Neware battery test system.D.W. and X.Y. designed and coordinated the experiments; D.W. and R.Y. carried out the experiment; D.W. and L.G prepared the experimental data; D.W. and X.W. wrote the paper.


V1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.Ca V1.1 functions as the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. This primary function of CaV1.1 depends on its physical interaction with the skeletal muscle ryanodine receptor (RyR1), but does not require calcium influx through the voltage-gated calcium channel itself. In fact, the classical adult CaV1.1a splice variant requires ∼30 mV stronger depolarization to activate its L-type calcium current (LTCC) than to activate EC coupling  myotubes or when CaV1.1a is expressed in heterologous cells—LTCC are barely detectable , which is the dominant CaV1.1 expressed in embryonic skeletal muscle . Bay K 8644 augments LTCC by shifting the fraction of total channel open events markedly to the long opening mode without altering the rate of activation  cell line GLT were cultured as previously described . At the 2, 10 HEPES, 0.1 Cs-EGTA, 2 Mg-ATP, and 0.2 Fluo-4 when calcium transients were recorded in parallel to calcium influx (pH 7.4 with CsOH). The extracellular bath solution contained (mM) 10 CaCl2, 145 tetraethylammonium chloride, 10 HEPES (pH 7.4 with tetraethylammonium hydroxide). To block the inward calcium currents, 0.5 mM Cd2+ and 0.2 mM La3+ (Cd2+/ La3+) were added to the bath solution. All recordings were made with an Axopatch 200A amplifier . Data acquisition and command potentials were controlled by pClamp software . Fluorescent calcium signals were recorded in parallel to patch clamp recordings using PTI photomultipliers . The voltage dependence of currents and calcium signals were analyzed as described in (μM): Bay K 8644: 1, Nifedipine: 0.1, 0.5, 5. When the highest concentration of DMSO (0.5%) was also applied to the bath solution in absence of DHPs, it had scarce effect on the calcium currents (IMax DMSO/IMax control = 0.8 ± 0.09) and voltage dependence (−5 ± 1 mV) of CaV1.1e.Patch pipettes  had resistance of 1.5–3 MΩ when filled with (mM) 145 Cs-aspartate, 2 MgClhold < 100 pA). To inactivate T-type currents, a 50 ms prepulse (to −50 mV) was applied before each sweep. Series resistance (Rs) was monitored between protocols, rejecting recordings with Rs drift > 20% or with Rs > 12 mΩ. After stable control recordings, the DHP-containing extracellular solution was applied at a flow rate of 1.5–2.5 ml/min, replacing at least twice the chamber volume. In these conditions, after 6 to 8 min of drug application, full drug effect was observed. Tail current amplitude was measured before and after Bay K 8644 application to quantify the changes in open probability (Po), assuming that no significant changes in the number of channels in the cell membrane occurred within 6–8 min of drug application. We have never observed a rundown of CaV1.1a or CaV1.1e in dysgenic myotubes.The effect of DHPs were tested in voltage-clamp experiments with a multiple-sweep protocol containing 200 ms long pulses from −50 mV to +80 mV in 10 mV increments before and after DHPs application. Between sweeps, the membrane voltage was kept at −80 mV  or the embryonic CaV1.1e (GFP-α1S-ΔE29) splice variants. Whole-cell voltage-clamp analysis of myotubes expressing one or the other splice variant revealed calcium currents with greatly distinct properties  and reached half-maximal activation at +35.1 ± 1.7 mV (V1.1e currents had five times larger current amplitudes (−10.4 ± 0.5 pA/pF) and a half-maximal activation at +6.4 ± 1.8 mV. During the 200 ms test pulse both currents showed little inactivation. Previously, we demonstrated that the different calcium current amplitudes are due to different biophysical properties of the two CaV1.1 splice variants and not to different expression levels , the peak current amplitude of Cav1.1a was increased more than fourfold (−9.2 ± 0.8 pA/pF). The half-maximal activation was left-shifted by 19.1 ± 1.5 mV (V1.1e (A–C). After Bay K 8644 application, the average peak current amplitude was increased by approximately twofold (−19.6 ± 1.5 pA/pF). The half-maximal activation was left-shifted by 9.3 ± 2.6 mV. The fractional inactivation measured at the end of the 200 ms test pulse to potential eliciting the maximal current amplitude was the same before and after the application of Bay K 8644 for both CaV1.1a (20.1 ± 0.03%) and CaV1.1e (22 ± 0.1%). Thus, CaV1.1e is sensitive to Bay K 8644 and the effect of exon 29 skipping and that of the channel agonist are partially additive. However, the effects of Bay K 8644 on Cav1.1a and Cav1.1e are quantitatively different. Bay K 8644 increased the maximal current (IMax) of Cav1.1a in a dose-dependent manner (D and E), at concentrations between 0.1 μM (approximately twofold) and 10 μM (∼4.5-fold). This did not occur for Cav1.1e, where the increase of IMax saturated (approximately twofold) at Bay K 8644 concentrations of 1 μM. The apparently weaker effects of Bay K 8644 on current amplitudes could be partially explained by the much larger leftward shift in the voltage dependence of activation induced by Bay K 8644 on CaV1.1a currents compared to CaV1.1e currents. To quantify the agonistic effect of Bay K 8644 at the same driving force we also examined the relative increase of tail currents in response to Bay K 8644 for both channel variants (D). The ratio of tail current amplitudes before and after application of Bay K 8644 is significantly larger (p = 0.0006) for Cav1.1a (7.1 ± 0.9) compared to Cav1.1e (2.7 ± 0.2). Together, these results show that indeed the effects of Bay K 8644 on voltage sensitivity and current amplitude in CaV1.1e are less than half the magnitude of those in CaV1.1a.Because the observed effects of deleting exon 29—increased current amplitude, left-shifted voltage-dependence of activation, but little change in the rate of activation—closely resemble the known effects of the current-enhancing gating modifier Bay K 8644 , we wond± 1.5 mV . AugmentV , which is in the same range that has previously been reported for Cav1.2 (B). The −5 mV shift of fractional activation at 0.5 μM nifedipine (C) is likely due to the strong reduction in current amplitude. Thus, the DHP nifedipine inhibits CaV1.1e calcium currents in skeletal myotubes in a concentration-dependent manner, like any other LTCC. Because LTCC through the adult CaV1.1a splice variant are already exceedingly small and functionally irrelevant in physiological EC coupling, we did not attempt to quantify the further reduction of CaV1.1a currents by nifedipine.Given our finding that Ca7 μM see D, whichr Cav1.2  and Cav1r Cav1.2  in similr Cav1.2 B. The −fedipine C is likV1.1e behaves similar to other L-type calcium channels. However, with respect to EC coupling it exhibits the distinctive skeletal muscle specific attributes: CaV1.1e can activate sarcoplasmic reticulum (SR) calcium release without calcium influx either at depolarizations to the reversal potential, or in the presence of the nonspecific calcium channel blocker Cd2+/La3+  also prevented the RyR1-dependent augmentation of LTCC   .V1.1e in skeletal muscle cells also requires direct interactions with the RyR1, we generated a CaV1.1e in which the II-III loop has been replaced by the corresponding but highly heterologous sequence of the Musca channel . When expressed in dysgenic myotubes CaV1.1e-SkLM was normally incorporated into peripheral junctions and developing triads, as indicated by its colocalization with RyR1 clusters at the cell surface (B). Thus, CaV1.1e-SkLM fulfills the basic structural condition for a functional interaction with the RyR1.To study whether the expression of the large calcium currents of Ca surface B. Thus,V1.1e or CaV1.1e-SkLM were analyzed in parallel with combined whole-cell patch-clamp measurements and fluorometric recordings of the fluorescent calcium indicator fluo-4. C shows representative traces of the cytoplasmic calcium transients (top) and calcium currents (bottom) at depolarizing steps to the maximally activating voltages. Plotting the voltage-dependence of peak calcium transients and calcium currents (D) shows that calcium transients and calcium currents of CaV1.1e and CaV1.1e-SkLM activate in parallel at the same voltages. Whereas at the reversal potential of +80 mV current densities decline to near zero, CaV1.1e calcium transients are still substantially activated, consistent with calcium influx-independent activation of SR calcium release . Calcium currents in myotubes expressing the chimeric CaV1.1e-SkLM channel are very similar in size and voltage-dependence to those of CaV1.1e. However CaV1.1e-SkLM calcium transients are greatly reduced in amplitude compared to those of CaV1.1e and they strongly decline at higher voltages, suggesting a strong dependence on the calcium influx. Application of 0.5 μM Cd2+/0.2 μM La3+ efficiently blocks the calcium currents at all voltages. In parallel calcium transients are abolished, indicating that the remnant calcium transients observed with CaV1.1e-SkLM are fully dependent on calcium currents. Thus, the small CaV1.1e-SkLM calcium transients arise from calcium influx through the CaV1.1 channel and possibly from calcium-induced calcium release from the SR . Eviden.1a-SkLM  and indiV1.1 is unique among the voltage-gated calcium channels in that it can activate SR calcium release independent of calcium influx by direct interactions with the RyR1. In the adult channel variant CaV1.1a this comes at the price of poor current properties. However, the embryonic CaV1.1e splice variant combines both skeletal muscle-type EC coupling properties with perfectly fine current properties. Here, we asked the questions of whether and how CaV1.1e calcium currents can be modulated; physiologically by retrograde interactions with the RyR1 and pharmacologically by DHPs. Our data show that 1) the current-enhancing effect of skipping exon 29 in CaV1.1e precludes further enhancement of the current by retrograde coupling, 2) the CaV1.1e currents can be further augmented by L-type current agonist Bay K 8644, and 3) blocked by L-type current antagonist nifedipine. These findings have important implications for understanding the molecular mechanisms regulating current activation and EC coupling in the skeletal muscle calcium channels, for understanding the function of the embryonic CaV1.1e splice variant in EC coupling, and for assessing its potential as drug target in myotonic dystrophy.The skeletal muscle CaV1.1-RyR1 interaction in CaV1.1e were eliminated, EC coupling failed but surprisingly the calcium currents were not affected. This finding is unexpected because CaV1.1a and CaV1.1e, which differ only with respect to the inclusion of 19 amino acids in an extracellular loop of CaV1.1a, interact with the RyR1 in the same way, and because earlier experiments with the adult splice variant CaV1.1a demonstrated that retrograde coupling is linked to orthograde coupling  by their dependence on the same critical sequences in the II-III loop  is in the same range as that reported for the CaV1.2, the major target for DHPs in smooth muscle cells, indicates that at the therapeutic dose side effects like hypotension would be expected.We further show that Cag et al.  demonstrpatients . TogetheV1.1e indicates that CaV1.1e LTCC are not subject to retrograde coupling with the RyR1 and that the physiological current modulation of CaV1.1a by retrograde coupling converges on the same molecular mechanism by which inclusion of the sequences encoded by exon 29 limits the size of the calcium current. On the other hand, our pharmacological characterization of CaV1.1e demonstrates that the channel agonist Bay K 8644 and inclusion of exon 29 use distinct molecular mechanisms for current enhancement. Its sensitivity to DHP calcium channel blockers identifies CaV1.1e as a potential drug target for the treatment of myotonic dystrophy.In conclusion, our biophysical analysis of the embryonic splice variant Ca


PCNA genes in their genome, but it has been reported that three PCNAsdo not always form a unique heterotrimer composed of one of each molecule. Thethermoacidophilic archaeon, Metallosphaera sedula, has three PCNAhomologue genes. Here, we demonstrated that the three PCNA homologues, MsePCNA1,MsePCNA2 and MsePCNA3, exclusively form a heterotrimer in a stepwise fashion;MsePCNA1 and MsePCNA2 form a heterodimer, and then MsePCNA3 binds to theheterodimer. We determined that the dissociation constants between MsePCNA1 andMsePCNA2, and between MsePCNA3 and the MsePCNA1:MsePCNA2 heterodimer are 0.29 and43 nM, respectively. Moreover, the MsePCNA1, MsePCNA2 and MsePCNA3heterotrimer stimulated M. sedula DNA ligase 1 activity, suggesting that theheterotrimer works as a DNA sliding clamp in the organism. The stable and stepwiseheterotrimerization of M. sedula PCNA homologues would be useful to generatefunctional protein-based materials such as artificial multi-enzyme complexes,functional hydrogels and protein fibres, which have recently been achieved byprotein self-assembly.Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role inDNA metabolism. Genome sequence analysis has revealed that some crenarchaea possessthree  DNA metabolism such as DNA replication and repair is a vital process for all organisms.In such processes, DNA sliding clamp, which is a ring-shaped protein with a centralcavity to encircle duplex DNA, plays a central role by recruiting DNA related proteinsfor accurate and efficient DNA replication and repair12Pyrococcusfuriosus DNA polymerase B forms two kinds of complexes with PCNA because of asecondary interaction in addition to the PIP-Box interaction, and consequently itsfunction is switched from polymerase to exonuclease by the configuration of thePCNA-enzyme complexArchaeoglobus fulgidus PCNA and RNaseH II, which removes RNA primersfrom Okazaki fragment junctions and cleaves misincorporated ribonucleotides, and changesthe orientation of the enzyme for DNA to access the substrate ribonucleotidesArchaea have PCNA as a sliding clamp, though the cellular structure is similar to that ofthe bacterial one1314151617PCNA genes. Theeuryarchaeon, Thermococcus kodakaraensis, and the crenarchaeon, Pyrobaclumaerophilum, possess two PCNAs. Two T. kodakaraensis PCNAs separately formhomotrimers and stimulate DNA polymerase activity, but only one is necessary for thevitality of the organismP.aerophilum strongly interacts with flap endonuclease, family 4 uracil DNAglycosylase and DNA polymerase B3, and therefore functions a PCNA, while the otherhomologue weakly interacts with the enzymesSulfolobus solfataricus, S. tokodaiiand Aeropyrum pernix, and are reported to form heterotrimers2324Eukaryotes have single PCNAs, but some archaea have multiple S. solfataricus PCNA is the first discovered heterotrimeric DNA sliding clamp. Thethree distinct PCNA subunits are monomeric proteins alone that form a heterotrimer in astepwise associationS. solfataricus PCNA subunitinteracts with specific DNA metabolising enzymes; SsoPCNA1 interacts with flapendonuclease 131PCNA genes are found in a genome. PCNAs from S.tokodaii and A. pernix, which form heterotimers, also form aheterotetramerin vitro, respectively. These findings indicate that the existence of threedistinct PCNA genes does not always indicate the formation of a uniqueheterotrimer composed of three distinct subunits.It is necessary to experimentally show the existence of exclusive heterotrimerisationeven if three distinct Metallosphaera sedula, which grows onelemental sulfur and metal sulphides at 50 to 80 °C and pH 2 to 3, andcan mobilise metals from metal sulfidesM. sedula heterotrimer ismore stable than the S. solfataricus PCNA heterotrimer. Furthermore, theheterotrimer stimulated the nick closing activity of DNA ligase 1, suggesting that theheterotrimer works as a sliding clamp. Recently, artificial protein self-assembly hasattracted great interest in biotechnology for developing functional materials such ashydrogels for cell stimulation37S. solfataricus PCNA subunits were promising scaffoldproteins to generate an artificial multi-enzyme complexM. sedula PCNA homologues thatform a stable heterotrimer in a stepwise manner would be useful for constructingfunctional protein-based materials.The thermoacidophilic crenarchaeon, S.solfataricus, which belongs to the order Sulfolobales, has three PCNAs,SsoPCNA1, SsoPCNA2 and SsoPCNA3, which are monomeric proteins that form aring-shaped heterotrimer in a stepwise manner; SsoPCNA1 and SsoPCNA2 form astable heterodimer, and then SsoPCNA3 associates with the heterodimer to form aheterotrimerA. pernix belongs to the orderDesulfurococcales and has three PCNAs, which form a heterotrimer, but one ofwhich forms a homotrimerA genome survey of crenarchaea revealed that each of the organisms belonging tothe orders Desulfurococcales, Sulfolobales, Acidilobales and Fervidicoccales hasthree distinct PCNA homologue genes, and each of those belonging to the orderThermoproteales has one or two homologue genes . S.solfM. sedula, Msed_0051 (UniProt: A4YCS6),Msed_1792 (UniProt: A4YHP5) and Msed_2250 (UniProt: A4YIY6), have the highestidentity with SsoPCNA1 (41%), SsoPCNA2 (53%) and SsoPCNA3 (46%), respectively phenomenon. In the subunits . The disS. solfataricusPCNA heterotrimer can be identified by the FRET between the fluorescent proteinsfused to the PCNAs (528/I477), which decreased upon addition of SsoPCNA2 analysis. MsePCNA3 did not interactwith the immobilised MsePCNA2 on the sensor chip, while MsePCNA1 rapidlyassociated with the immobilised MsePCNA2 on the sensor chip and dissociatedextremely slowly (Kd) ofMsePCNA1 and MsePCNA2 was 0.33 ± 0.12 nM , which was previously determined to be2.7 × 102 nM using a GST-fusedSsoPCNA1-immobilised sensor chipM. sedula PCNA homologues , MseLig1, has 69% sequence identity with S.solfataricus DNA ligase 1, which is known to be stimulated in thepresence of the S. solfataricus PCNA heterotrimerM. sedula PCNAhomologues together  of a heterodimer, themixtures containing MsePCNA1 and SsoPCNA2 in molar ratios of 1:1, 2:1 and 1:2were analysed by size exclusion chromatography .The equKd values of the M. sedula PCNA homologues and theS. solfataricus PCNAs were determined by SPR analysis. SsoPCNA1dissociated from the immobilised MsePCNA2 faster than MsePCNA1, and as a resultthe Kd value between SsoPCNA1 and MsePCNA2 was higher thanthat between MsePCNA1 and MsePCNA2  in a 20 mMpotassium phosphate buffer, pH 7.4, containing 150 mM KCl and10 mM imidazole was incubated on ice for 30 min. Next, the resinwas washed twice with 1 ml of the buffer. The proteins tethered to theresin were eluted with 30 μl of the 20 mM potassiumphosphate buffer, pH 7.4, containing 150 mM KCl and 500 mMimidazole. The eluted proteins were separated by SDS-PAGE and stained byCoomassie Brilliant Blue R-250.A suspension prepared from 30 μl of a 50% slurry of TALON Metalaffinity resin , and 60 μl of a mixturecontaining 30 μM HisA mixture containing 120 μM protein(s) in 150 μlof 50 mM potassium phosphate buffer, pH7.4, containing 150 mMKCl was incubated on ice for more than 1 h. Then, the mixture wassubjected to size exclusion chromatography on Superdex 200 10/300 GL column  at a flow rate of 1.0 ml/min with50 mM potassium phosphate buffer, pH 7.4, containing 150 mMKCl.−1 cm−1,respectively, at 280 nm, which were calculated from their amino acidcompositions. Those of YPet-fusion proteins and CyPet-fusion proteins weredetermined spectrophotometrically, using the extinction coefficients of YPet at514 nm(ε514 = 104 mM−1cm−1) and CyPet at 433 nm(ε433 = 35 mM−1cm−1), respectivelyProtein concentrations of SsoPCNA1, SsoPCNA2, SsoPCNA3, MsePCNA1, MsePCNA2 andMsePCNA3 were determined spectrophotometrically, using the extinctioncoefficients of 14.4, 13.4, 13.4, 17.4, 11.9,8.94 mMAccording to the Biacore  protocols, about 350 RU of MsePCNA2 orSsoPCNA2 were immobilised on Sensor Chip CM5. Then, the sensorgram was monitoredby injecting various concentrations of MsePCNA1 or SsoPCNA1. At the end of eachcycle bound proteins were removed by washing with 10 mM glycine-HClbuffer, pH 1.5, at a rate of 10 ml/min for quantitative analysis and ata rate of 20 μl/min for kinetics analysis. The kinetics valueswere determined from association and dissociation curves of the sensorgrams,using BIAevaluation software. To determine the dissociation constants ofMsePCNA3 and SsoPCNA3 from protein complexes formed on the sensor chip, variousconcentrations of MsePCNA3 or SsoPCNA3 were injected following application of1 μM MsePCNA1 or SsoPCNA1. To eliminate the effect of thedissociation of MsePCNA1 or SsoPCNA1, the sensorgram obtained without injectingMsePCNA3 or SsoPCNA3 was subtracted from each sensorgram. As a result, theeffective increase in the SPR signal by the binding of MsePCNA3 or SsoPCNA3 to aprotein complex coupled to the sensor chip was determined. The dissociationconstants were determined by plotting the increase in the SPR signal against theinjected protein concentration followed by fitting response to Langmuiradsorption isotherm using Prism6 . All themeasurements were performed in 10 mM HEPES-HCl buffer, pH 7.4, with150 mM NaCl.M. sedula DNA ligase 1,1 μM M. sedula PCNA homologues, 1 μM 5′phosphorylated DNA (5′-CAGAGGATTGTTGACCGGCCCGTTTGTCAG-3′), 1μM 5′ hexachlorofluorescein (HEX)-labelled DNA(5′-CGCACCGTGACGCCAAGCTTGCATTCCTACAGGTCGACTC-3′) and1 μM template DNA(5′-CGTTGCTGACAAACGGGCCGGTCAACAATCCTCTGGAGTCGACCTGTAGGAATGCAAGCTTGGCGTCACGGTGCGCCAAC-3′)was incubated at 50 °C for 90 min. Then,10 μl of loading solution containing 98% formamide,10 mM EDTA, bromophenol blue and xylen cyanol were added to10 μl of the reaction mixture. After boiling for 5 min,6 μl of the mixture were separated on 10% 8 M-ureapolyacrylamide gel.A mixture containing 10 mM HEPES, pH 8.0, 10 mM magnesiumchloride, 1 mM ATP, 300 nM How to cite this article: Iwata, F. et al. Three proliferating cellnuclear antigen homologues from Metallosphaera sedula form a head-to-tailheterotrimer. Sci. Rep.6, 26588; doi: 10.1038/srep26588 (2016).