A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels . The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.Intracellular Ca Junctional membrane complexes between the plasma membrane and the ER/SR are needed to provide effective mechanisms for cross talk between Ca2+ channels/transporters in the plasma membrane and Ca2+-release channels in intracellular membranes (3) receptors have a direct association, and IP3 via its receptors potentiates TRPV4 sensitivity to the mechano- and osmo-transducing messenger 5′-6′-epoxyeicosatrienoic acid   . The plaubregion . This exand NCX3 . The expand NCX3 . The resh muscle . For exaterioles , and NCX stomach . The phy stomach  provide 3 receptor (IP3R), and NCX and the potential functions of such an interaction in ASM contraction. The discovery of such an interaction would further understand ASM tension regulation and may contribute to the development of compounds effective in the treatment of respiratory diseases, especially tracheal spasm.To rapidly and efficiently activate downstream signaling molecules, several molecules involved in the same signal transduction form signal complexes. In the present study, we evaluated whether an interaction occurred for TRPV4, the IP3R1 antibody, and KB-R7943 were purchased from Santa Cruz Biotechnology. The anti-NCX1 antibody, anti-NCX2 antibody, and anti-NCX3 antibody were purchased from the Beijing Bioss company.Carbachol, GSK1016970A, KB-R7943, HC067047, apamin, and lithium chloride (LiCl) were purchased from Sigma (United States). Iberiotoxin (IbTX), and TRAM34 were purchased from MedChemExpress. The anti-TRPV4 antibody, anti-IPad libitum. The equations should be inserted in editable format from the equation editor.Male Kunming mice (18–20 g) were obtained from the Experimental Animal Center of Anhui Medical University and used in accordance with guidelines of the U.S. National Institutes of Health (NIH publication No. 8523). This study was approved by the Animal Experimentation Ethics Committee of Anhui Medical University. Mice were housed in a facility with a 12-h light-dark cycle, a temperature of 24°C, and humidity of 60% and were allowed food and water 3 25.2, KCl 4.7, CaCl2 2.5, glucose 11.1, KH2PO4 1.2, and MgSO4.7H2O 1.2). After the epithelial layer was rubbed off, the trachea was digested with 0.2% collagenase type IA and 0.9% papain for 60 h. The dispersed tracheal smooth muscle cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 μg/mL streptomycin and 100 U/mL penicillin at 37°C. In order to remove non-adherent cells, the medium was replaced after a 60 h incubation. The remaining adherent ASM cells were cultured at 37°C with 5% CO2 for 5–7 days before experimental use. The ASM marker α-SMA and fibroblasts marker FSP are used to characterize the specificity of ASM cells , and then incubated with primary antibodies against TRPV4  followed by incubation with a goat anti-rabbit secondary antibody. Images were acquired by microscopy .2+]i was measured as previously described (2+ binding dye) and 0.02% pluronic F-127 (Invitrogen) for 30 min. The cells were treated with 1 nM GSK1016790A with or without 10 μM HC067047. Fluorescence was recorded using a fluorescence microscope  having a xenon lamp with excitation and emission wavelengths of 488 nm. The [Ca2+]i changes are presented as the ratio of the experimental fluorescence relative to the initial fluorescence intensity (F1/F0).The i in primary cultured mouse ASM cells, and this response was completely abolished by HC067047 (10 μM), a TRPV4 blocker . Each forms a Ca2+ channel that is co-regulated by IP3 and Ca2+, but the subtypes differ in their expression patterns  (4 μM) or the IP3 receptor inhibitor heparin (150 μg/mL), but could not inhibit by raynodine receptor (RYR) inhibitor raynodine (10 μM)  . These r2+ increase resulted in relaxation of tracheal rings that were precontracted with 1 mM carbachol, suggesting that activation of TRPV4 may excite different downstream signaling pathways dependent on the local Ca2+ concentration. NCX has an important role in regulating intracellular Ca2+ homeostasis. In order to assess whether NCX is activated by the TRPV4-induced increase in local Ca2+ concentration, we used the specific NCX inhibitor KB-R7943 (5 μM) and Krebs–Henseleit solution that NaCl was substituted by LiCl (118 mM). We found that compared with the response in the control group, pretreatment with 5 μM KB-R7943 or with LiCl significantly inhibited the GSK1016790A-induced tracheal ring relaxation  is another potential mechanism for Ca2+-mediated relaxation in ASM cells. To determine whether the observed relaxation was due to TRPV4-mediated Ca2+ activation of BKCa, we pretreated mouse tracheal rings with the specific BKCa blocker IbTX. Our results showed that the pretreatment of IbTX (50 μM), even in the presence of the intermediate-conductance Ca2+-activated K+ (IK) channel blocker TRAM34 (10 μM) and the small-conductance Ca2+-activated K+ (SK) channel blocker apamin (1 μM), had no effect on the TRPV4-induced relaxation of carbachol-induced precontracted tracheal rings  and TRPV4 inhibitor HC067047(10 μM) to mice and measure the breath rate of mice at 0, 30, 60, and 90 min of aerosol inhalation. The results showed that TRPV4-specific inhibitor HC067047 significantly increased the respiratory rate and the TRPV4 activator GSK1016790A significantly decreased the respiratory rate in time dependence  Although the specific TRPV4 agonist GSK1016790A induced a transient [Ca2+]i rise in primary cultured mouse ASM cells, this agonist also caused a concentration-dependent relaxation in mouse tracheal rings precontracted by carbachol. (2) Inhalation of TRPV4 blocker increased the respiratory rate of mice, whereas inhalation of the TRPV4 agonist decreased the rate. (3) The specific NCX inhibitor KB-R7943 and LiCl significantly inhibited the GSK1016790A-induced relaxation of tracheal rings precontracted with carbachol. (4) Pretreatment with the BKCa blocker IbTX, the IK blocker TRAM34, and the SK blocker apamin had no effect on the TRPV4-induced relaxation of tracheal rings precontracted with carbachol. (5) Co-IP assay analyses suggested that TRPV4, IP3R1, and NCX form a complex in ASM.The major finding of this study was that TRPV4, IP2+]i rise largely depends on the balance between Ca2+ releasing from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. In ASM cells, carbachol binds with GPCRs and mediates activation of phospholipase C to trigger IP3 production and IP3-mediated Ca2+ release from the SR (3R in the SR is critical for the Ca2+ rise induced by carbachol. The junctional membrane complexes between the plasma membrane and the ER/SR are needed to provide effective mechanisms for cross talk between Ca2+ channels/transporters in the plasma membrane and Ca2+-release channels in the intracellular membranes (3 receptors have a direct association, and IP3 via its receptors potentiates TRPV4 sensitivity to the mechano- and osmo-transducing messenger 5′-6′-epoxyeicosatrienoic acid (3R1 physically interact in the mouse ASM cells. These results suggested that carbachol binds with GPCRs and induces IP3-mediated Ca2+ release from the SR, and TRPV4 in the plasma membrane is activated through the physical interaction with the IP3R in mice ASM cells.The contraction of ASM cells are modulated by the rapid i, activation of TRPV4 caused an additional local Ca2+ rise, and this lead to the relaxation of the ASM which is blocked by the NCX inhibitor KB-R7943 or LiCl substituting NaCl in Krebs–Henseleit solution. The results of previous studies examining the functional roles of NCX in ASM appear conflicting, suggesting that the contribution of NCX in ASM may be species dependent. Among the three isoforms of NCX, NCX1 is expressed in human ASM and NCX1-mediated Ca2+ fluxes contribute to enhanced [Ca2+]i regulation in airway inflammation (Naeostasis . The platraction . Our datammation . Our res3R1 physically interact with NCX1,2,3. These findings support the physical interaction of NCX, TRPV4, and IP3R1 between the plasma membrane and the SR membrane which induced that the local Ca2+ rise induced by TRPV4 actives rapidly the adjacent NCX and then lead to a Ca2+ efflux, and form a negative feedback to elicit trachea relaxation. We also tested whether Ca2+-activated K+ channels, including BKCa, IK, and SK, played a role by using the specific blockers IbTX, TRAM34, and apamin, respectively. Our results showed that Ca2+-activated K+ channels do not participate in the TRPV4-mediated relaxation of tracheal rings precontracted by carbachol.In order to rapidly and efficiently activate downstream signaling molecules, several molecules involved in the same signal transduction may form signaling complexes. Regarding the subcellular distribution of NCX, the physical proximity of NCX to the perimembranous SR in vascular smooth muscle has been demonstrated, and other investigators have suggested that a similar situation occurs in ASM . A poten3R1 with NCX as a novel regulatory mechanism underlying TRPV4-mediated Ca2+ signaling (3-mediated Ca2+ release from the SR, and TRPV4 in the plasma membrane is activated through the physical interaction with the IP3R. The local Ca2+ rise induced by TRPV4 actives the adjacent NCX to elicit to a Ca2+ efflux and relaxation of ASM cells. This TRPV4-IP3R1-NCX functional interaction achieves rapid signal transmission and regulation and contributes to ASM tension. This interaction suggests a potential target for regulation of ASM tension and treatment of certain respiratory diseases, especially tracheal spasm.We suggest a physical and functional interaction of TRPV4 and IPignaling . CarbachThe raw data supporting the conclusion of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of Anhui Medical University.JZ, YW, SB, SD, HG, SY, SC, JL, and HW developed the methodological aspects of analysis and data interpretation. JZ and SD analyzed the data, prepared the figures, and drafted the manuscript. JD drafted the manuscript. YW, SB, and HG contributed the extra experiments and followed reviewer’s suggestions. All authors revised the manuscript, approved the final version, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since HddC is a potential new target to develop antibiotics, the analysis of the structural and functional relationship of the complex structure will lead to a better idea to design inhibitory compounds. X-ray crystallography and biochemical experiments to elucidate the guanine preference were performed based on the multiple sequence alignment. The crystal structure of HddC from Yersinia pseudotuberculosis (YPT) complexed with guanosine 5′-(β-amino)-diphosphate (GMPPN) has been determined at 1.55 Å resolution. Meanwhile, the mutants revealed their reduced guanine affinity, instead of acquiring noticeable pyrimidine affinity. The complex crystal structure revealed that GMPPN is docked in the catalytic site with the aid of Glu80 positioning on the conserved motif EXXPLGTGGA. In the HddC family, this motif is expected to recruit nucleotides through interacting with bases. The crystal structure shows that oxygen atoms of Glu80 forming two hydrogen bonds play a critical role in interaction with two nitrogen atoms of the guanine base of GMPPN. Interestingly, the binding of GMPPN induced the formation of an oxyanion hole-like conformation on the L(S/A/G)X(S/G) motif and consequently influenced on inducing a conformational shift of the region around Ser55. Yersinia pseudotuberculosis (YPT) is a Gram-negative, rod-shaped, enteropathogenic bacteria highly relevant to Yersinia pestis (YP) and Yersinia enterocolitica (YE) [YPT causes self-limiting, ileitis, and mesenteric lymphadenitis in humans and spreads through the fecal-oral route [YPT infection outbreaks sporadically in various parts of the world, including North America, Europe, Russia, and Japan [YE [YPT can lead to intestinal necrosis, hemorrhage, multi-organ abscesses, or bacteremia [YPT show the one of sequelae like reactive polyarthritis that can carry on for weeks to months [ica (YE) . YPT caual route ,2. Humannd Japan . The disapan [YE . Clinicao months .Escherichia coli (E. coli), Pseudomonas, and Yersinia. An outer membrane permeability barrier is vital to the antimicrobial resistance of these bacteria because they protect peptidoglycan and other intracellular targets from various antibiotics [Lipopolysaccharide (LPS) is a characteristic outer barrier of microorganisms and contributes to self-defense, membrane integrity and permeability critical for bacterial survival . In partibiotics . LPS is ibiotics .manno-heptose and its derivatives participating as building blocks of the core region are essential components of LPS in most Gram-negative bacteria. d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme involved in the heptose biosynthetic pathway and produces GDP-d-glycero-α-d-manno-heptose, an activated form of monosaccharides acting as glycosyl donors in glycosylation reactions. Therefore, HddC is a good target for developing antibiotics or adjuvants. Recently, the first crystal structure of the HddC family has been determined [YPT (YpHddC) and is the target for against yersiniosis. Its X-ray crystal structure revealed remarkable structural similarity to those of cytidylyltransferase and uridylyltransferase. Nevertheless, YpHddC uses guanosine 5′-triphosphate (GTP) for catalysis rather than cytidine 5′-triphosphate (CTP) and uridine 5′-triphosphate (UTP). Despite the interesting difference, the understanding of the inclination for GTP is limited due to the absence of its crystal structure complexed with GTP. Here, we report the crystal structure of YpHddC complexed with guanosine-5′-(β-amino)-diphosphate (GMPPN), a GTP analogue, to elucidate the structure-function relationship of YpHddC. Based on the crystal structural data, we found a key residue to interact with GMPPN. Then, a mutation of the key residue was performed to see if the base preference would be changed.The carbohydrate glycero-termined . It was YpHddC was concentrated to 12 mg·mL−1. The final concentrated native YpHddC and mutants seemed to be nearly 99% pure at around 26 kDa on SDS-PAGE. The initial crystallization screening was done with the native protein bound Guanosine 5′- triphosphate (GMPPNP). Crystals of the native YpHddC protein with GMPPNP appeared with long rectangular shapes in 0.05 M 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) pH 7.5, 0.2 M magnesium chloride, 1.2 M sodium citrate tribasic dehydrate. Crystallization information is summarized in Purified native 3, with unit cell parameters a, b = 80.526 Å, c = 53.081 Å. Based on the Matthews coefficient [M = 3.11 Å3 Da−1). Details of the data-collection statistics of native YpHddC with GMPPN is presented in YpHddC were visible and the model of YpHddC was also well generated. The extra densities were well matched with one magnesium, one GMPPN  was the best cryoprotectant in this case. X-ray diffraction data were collected at a single wavelength λ = 0.97928 Å). The crystal belongs to the primitive tetragonal space group P47928 Å. Tne GMPPN , and twoYpHddC complexed with GMPPN revealed that its overall structure is similar to that of the UDPGP family (IPR002618) classified by InterPro [N-acetylglucosamine pyrophosphorylases (EC: 2.7.7.23) and UDP-N-acetylhexosamine pyrophosphorylases. In the protein family hierarchy, the UDPGP family can be classified as the subset of the homologous superfamily, nucleotide-diphospho-sugar transferases [YpHddC is structurally and functionally related to GalU. This family shares a unique topology of an extended β-sheet composed of nine β-strands sandwiched by several α-helices. This topology comprises a common sugar nucleotidylyltransferase (SNT) fold carrying the canonical signature motif GXGXR (10GLGTR14 in YpHddC) for nucleotide-binding [Burkholderia vietnamiensis , there are two insertions: one α-helix is attached to the C-terminus (most frequently found in other members) and extra two α-helices are inserted into the loop between α2 and β3. In another case of mannose-1-phosphate geranyltransferase from Thermus thermophilus HB8 (PDB ID: 2CU2), two α-helices are inserted at the loop between 310-helix G2 and π-helix π1. It is noteworthy that there is no insertion in these places in the case of YpHddC. However, YpHddC shows unique local conformations in some regions. Compared with its closest homologs, it has the very simple two loops between β6 and β7 and between β8 and β9. In addition, it has the longest loop connecting β1 and α1  was prepared because its active pocket is much more similar to that of the UDPGP family. An S53N mutant was also made in order to copy the active site of PpMurU. With these mutants, the malachite green assay has been performed. Their activity was measured and represented as a bar graph (The three mutants were designed and cloned to elucidate the base preference for ar graph . Brieflyar graph .There was a clear electron density that turned out to be GMPPN in the GTP binding pocket. In the protein data bank (PDB), no GMPPN molecule is searched. Therefore, in our knowledge, it is the first GMPPN structure determined by X-ray crystallography. In contrast, some of AMPPN bound structures can be searched in the PDB . The cavity is mainly composed of β1, β2, β3, and α3 together with their connecting loops L2, L4, L6 .The residues on the loop L2 are Leu7, Ala8, Gly9, Gly10, Leu11, and Gly12 a. manno-octulsonic acid synthetase [They are contributing to building the canonical signature motif, GXGXR. Ala8, Gly9, and Gly10 experience an induced-fit conformational change when they interact with GMPPN in the crystal structure. In detail, an oxyanion hole-like conformation was induced by the three rearranged backbone nitrogen atoms. Consequently, the induced oxyanion hole attracts the O2′ oxygen atom of the ribose moiety of GMPPN c. The dinthetase .YpHddC. Interestingly, this serine residue of YpHddC does not show meaningful interaction with the guanine base. Actually, the atomic positions of Ser53 and Leu54 are unaffected after the GMPPN binding  and PpMurU of which the native and pyrimidine-bound forms have been published. The volume of each cavity for bases is around 290 Å3 regardless of bases when calculated with the MOLCAD module implemented in SYBYL-X 2.1.1. [The residues on the loop L6 are Glu80, Asn81, Glu82, Pro83, Leu84, and Gly85 c. Of theX 2.1.1.  ThereforYpHddC [StRfbF [L6 contains Glu80 which is a key residue to interact with GMPPN. Two oxygens of the Glu80, Oε1, and Oε2, make interactions with N2 and N1 atoms of guanine with the distances of 2.84 Å and 2.79 Å, respectively c. Previo [StRfbF .YpHddC confirmed its GTP preference and also revealed that Glu80 is the key residue to hold GTP in the nucleotide-binding pocket. In contrast, uridylytransferases use a glutamine residue in their active site to recruit UTP as observed in BvGalU. In the X-ray crystal structure of BvGalU, the Nε2 and Oε1 atoms of Gln105 play a critical role in recruiting UTP. The two atoms of Gln105 make electrostatic interactions with the O4 and N3 atoms of bound UDP-glucose in the distances of 2.85 Å and 3.73 Å, respectively. Majority of the crystal structures of uridylytransferases currently deposited in the PDB confirm the presence of a glutamine residue in their nucleotide-binding site to recruit UTP.The GMPPN complexed structure of YpHddC to Gln80 has been performed to check whether a single point mutation would be enough to alter the guanine preference. Unexpectedly, the E80Q mutant augmented the inclination of adenine preference rather than uridine  because the top structural homolog of s PpMurU . The cryactivity . These rYphddC gene coding for the YpHddC protein (NCBI Reference Sequence: WP_050090753.1) was inserted in the pBT7 plasmid DNA (pDNA) and gained from Bioneer . To express the protein, we transformed pDNA into E. coli BL21 (DE3). Transformed E. coli cells were incubated on Luria-Bertani (LB) agar plates. Several colonies were chosen and grown in test tubes with a cap to determine the condition for culturing in bulk. In the process, a cell stock was prepared and frozen. The mass culture proceeded at 310 K with shaking. As the absorbance at 600 nm of broth reached 0.6–0.8, Isopropyl-β-d-1-thiogalactopyranoside was put for expression of YpHddC. The culture was placed back into an incubator shaker and incubated at 310 K for 3 h. To collect cells, we centrifuged the culture fluid at 7650 g (6500 rev·min−1) for 10 min in a high-speed refrigerated centrifuge at 277 K. The cultured cell pellet was suspended and fragmentized using a Digital Sonifier 450 . Cell debris was pelleted by centrifugation. The supernatant was affinity-purified using a HisTrap column on an ÄKTA explorer system  based on the previous study [YpHddC protein containing an N-terminal His6-tag followed by 8 amino acid sequence, EFSQQDSD (MHHHHHH EFSQQDSD) was concentrated.The YpHddC combined with GTP analog, GMPPNP. The screening was carried out at room temperature using the sparse-matrix method [YpHddC combined with 2.25 mM GMPPNP and 10 mM MgCl2 were obtained in 0.05 M HEPES pH 7.5, 1.4 M sodium citrate tribasic dihydrate. To obtain high-quality co-crystals of native YpHddC combined with GMPPNP, we carried out fine refinement around the condition of 0.05 M HEPES pH 7.5, 1.4 M sodium citrate tribasic dehydrate with 0.01–0.2 M magnesium chloride. Magnesium is absolutely required for catalytic activity of YpHddC as a cofactor and also was essential for crystallization. Finally, co-crystals of YpHddC with GMPPN, having good quality for diffraction, were obtained in 0.05 M HEPES pH 7.5, 0.2 M magnesium chloride, 1.2 M sodium citrate tribasic dehydrate.Screening for crystallization conditions was performed with native x method  with sevCrystals were soaked in LV CryoOil  as a cryoprotectant and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at a single wavelength on beamline 11 C at Pohang Light Source using a Pilatus 6 M detector  placed 300 mm from the sample . The oscYpHddC (PDB ID: 5XHW) as a search model. Iterative density modification, model building, and refinement were performed using the AutoBuild wizard of PHENIX. The model refinement together with the addition of water and bound molecules were performed using Crystallographic Object-Oriented Toolkit (COOT) [Molecular replacement (MR) was performed using PHENIX  with thet (COOT)  and PythYphddC gene was used as a template for constructing two single  mutants. The sequences of the mutagenic primers of S53N mutant YphddC were 5′-GGGCGTGCAGCGCATTATTCTGAACCTGAGCTATAA-3′ for the forward strand and 5′-TTATAGCTCAGGTTCAGAATAATGCGCTGCACGCCC-3′ for the reverse strand. The sequences of the mutagenic primers of E80Q mutant YphddC were 5′-GCCCAGCGGTTCGTTCTGAATAATAAAATCCACTTTGCTGTTCACC-3′ for the forward strand and 5′-GGTGAACAGCAAAGTGGATTTTATTATTCAGAACGAAC CGCTGGGC-3′ for the reverse strand. YphddC mutated Glu80 to Gln80 was used as a template for constructing one double (S53V/E80Q) mutant. The sequences of the mutagenic primers of S53V/E80Q mutant YphddC were 5′-TCCGCTTTATAGCTCAGGACCAGAATAATGCGCTGCACG-3′ for the forward strand and 5′-CGTGCAGCGCATTATTCTGGTCCTGAGCTATAAAGCGGA-3′ for the reverse strand.Synthesized pBT7 plasmid DNA (pDNA) containing E. coli XL10-Gold competent cells were grown in 500 µL NZ amine - yeast extract (NZY) broth and were incubated at 310 K for an hour. A 100 µL aliquot of the NZY broth was spread onto LB plates containing 50 mg·ml−1 ampicillin. Transformed mutant plasmids were purified using DNA-spin plasmid DNA purification kit .The polymerase chain reaction (PCR) and transformation were performed with the QuikChange Lightning Site-Directed Mutagenesis Kit  as stated by the manufacturer’s protocol. The transformed YphddC was constructed and used as a template for mutagenesis. The expression plasmids including the S53N mutant, the E80Q mutant, and the S53V/E80Q mutant, constructed using the Mutagenesis Kit, were isolated. DNA sequences were checked by gene sequencing through GenoTech corp.  and those recombinant plasmids were transformed into E. coli BL21 (DE3). The mutant proteins were purified using the same protocols used in the wild type.Gene synthesis, expression, and purification of the protein was fulfilled using the same method that was published previously . The expYpHddC and its mutant. The GMP moiety from GTP is transferred to d-glycero-α-d-manno-heptose-1-phosphate by YpHddC. Consequently, GDP-d-glycero-α-d-manno-heptose and pyrophosphate (PPi) are released. Instead of d-glycero-α-d-manno-heptose-1-phosphate, we used α-d-Mannose-1-phosphate (αM1P) purchased from Sigma for a substrate. PPi was degraded by inorganic pyrophosphatase (IPP) and detected using the malachite green reagent [w/v) ammonium molybdate ((NH4)6Mo7O24) in 4 N hydrochloride, 1% (v/v) Tween 20 and 0.045% (w/v) malachite green solution in the ratio 1:3:0.1 and stirring for 30 min at room temperature (RT). The detection reagent was filtered with a 0.2 μm PVDF syringe filter  and stood for 1 h at RT before use. For NTP reaction study, reaction solutions were containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.04 unit IPP and HddC (0.05 mg·ml−1). In addition, substrates of YpHddC and its mutants, NTP (0.25 mM) and αM1P (1 mM), were used. After incubation at 25 °C for an hour, 40 μL of reaction mixtures were mixed with 160 μL of the color reagent. The mixtures were waited for 15 min to react. The absorbance of the final mixtures was measured at the specific wavelength (660 nm) using a microplate spectrophotometer . The comparative bar graph was worked with the GraphPad Prism 7.03 .A Malachite green assay method with pyrophosphatase  was used reagent . A detecYpHddC is its GTP preference regardless of overall structural similarity to the UDPGP family. Glu80 of YpHddC is the key residue for the GTP affinity as revealed by E80Q and S53V/E80Q mutants. Other minor conformational changes seem to be important for nucleotidylyltransferase activity. The continuous intuitive mutant experiments based on the multiple sequence alignment together with the active site structural comparison were carried out to endow pyrimidine affinity for YpHddC. Instead of discovering key residues for pyrimidine affinity, however, the results could confirm the importance of Ser53 and Glu80 of YpHddC for its GTP preference. All mutants indicated that key residues holding different bases in nucleotidylyltransferase seem to be very unique and thus their base preference could not be easily copied. A further mutational and functional study is going on to alter the GTP affinity of YpHddC.In summary, the first X-ray crystal structure of GMPPN bound from among the HddC family was determined. The main molecular property of


During these early postnatal days, intercellular propagating Ca2+ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca2+ APs independently or require a trigger by an ISC Ca2+ wave. To identify the Ca2+ transients of IHCs underlying Ca2+ APs and to analyze their dependence on ISC Ca2+ waves, we performed fast Ca2+ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca2+ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca2+ waves in ISCs. ISC Ca2+ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca2+ elevations of small amplitude appeared to be triggered by ISC Ca2+ waves in IHCs of Cav1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca2+ influx through Cav1.3 channels. The Ca2+ wave-triggered Ca2+ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca2+ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca2+ elevations in adjacent ISCs. PPADS irreversibly abolished Ca2+ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca2+ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2–5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca2+ waves. Neonatal IHCs thus spontaneously generate electrical and Ca2+ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker’s organ indicating two sources of spontaneous activity in the developing auditory system.Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca These APs translate into release of glutamate  generate Ca2+ APs autonomously  as a trigger  ATP-induced depolarization to the threshold for regenerative APs  and P2X2R/P2X3R double knockout mice  in accordance with the German law on the use of laboratory animals and were approved by the Saxonian District Government, Leipzig (TVV59/16 and T34/16), and the regional board for scientific animal experiments of the Saarland. Prehearing mice (P4–P6) of either sex were studied. NMRI mice  were used unless stated otherwise and are designated as controls. Ca2, 0.9 MgCl2, 5.6 glucose, 10 HEPES and 0.9 NaH2PO4, 305 mOsm/l, pH 7.35. The organ of Corti was dissected out of the cochlea with great care to preserve the integrity of the tissue. The apical turn was cut and placed in a recording chamber underneath a soft thread of a nylon mesh, which was attached to a steel ring  that had been dissolved in a Pluronic-DMSO mixture . To allow for uptake of Fluo-8 AM and hydrolysis, the recording chamber with the tissue was kept in a dark, humid plastic box for 40 min at room temperature. The chamber was mounted on the stage of the upright laser scanning microscope LSM 710  and perfused with perilymph-like solution for at least 7 min to remove any Fluo-8 AM prior to the experiment. Only healthy looking epithelia with smooth surfaces, absence of vacuoles and low background fluorescence in both IHCs and ISCs were used for imaging : 143 NaCl, 5.8 KCl, 2.2 MgCl2, 5.6 glucose, 10 HEPES, 0.9 NaH2PO4 and 0.5 EGTA acid, 305 mOsm/l, pH 7.35. The distance between the scan field and the lateral edges of the tissue combined with the application of perilymph-like solution without/with agonist/antagonists from the pillar side prevented spillover of ATP from potentially damaged cells and ISCs. The delay in solution flow from reaching the IHCs to reaching the ISCs was estimated to be <20 ms.Neonatal mice were sacrificed by decapitation. Cochleae were immediately dissected in ice-cold physiological solution mimicking perilymph (in mM): 143 NaCl, 5.8 KCl, 1.3 CaClg Figure . IHCs weg Figure . Agonist2+ indicator with a high quantum yield and a Kd of 389 nM we determined the relative fluorescence changes 2+]i changes in ISCs and IHCs off-line:Data were recorded using a LSM 710 based on an upright AxioExaminer microscope with 40×/1.0 NA or 20×/1.0 NA Plan Apochromat water immersion objectives and Zen-2012 SP1 (Black edition) software . Fluo-8 fluorescence was excited with the 488 nm line of an argon laser. Laser intensity was set to very low levels to allow for long continuous recordings  while avoiding photobleaching and strong phototoxicity. We opened up the pinhole to maximal seven airy units accepting a reduced spatial resolution in favor of increased fluorescence intensity at the chosen low laser power and a higher scan speed. Recordings were performed at room temperature (21°C ± 1°C). Because Fluo-8 based on Fluo-4  is the fluorescence intensity of a pixel at a given time t and F0 the fluorescence of this pixel averaged over the first 10 images without obvious Ca2+ changes.where 2+ waves in Kölliker’s organ, scan fields of about 200 μm × 100 μm were chosen resulting in frame rates of ≤5 Hz (scan times >200 ms). To resolve the much faster fCaTs in IHCs, we increased the frame rate by: (i) imaging smaller scan fields; and (ii) increasing the pixel size to 0.4 μm × 0.4 μm (up to 0.6 μm × 0.6 μm). Scan fields were 140 μm ± 21 μm in width  and 27 μm ± 5 μm in height . With these settings, photobleaching amounted to 0.5 ΔF/F0 at maximum during periods of ≥6 min. Offline analysis and graphical representation was performed using FIJI  of fluorescence images were defined by activity patterns within IHCs and ISCs and were validated using a high-resolution transmitted light image acquired prior to the t-series with a 100-fold smaller pixel area. Transmitted light images were also taken after a t-series to check for a potential tissue drift. Data are reported as mean ± standard deviation (SD) except data in Figure For imaging the spread of ISC Ca Figures  and comp2+ waves and corresponding waves of light refraction (crenation waves) in the region of ISCs. This is consistent with previous studies of ISCs of Kölliker’s organ in the immature organ of Corti  to Ca2+ waves extending over various distances. Small Ca2+ waves propagated radially to maximum two neighboring ISC circles comprising about 200–600 μm2, whereas large waves covered areas from 600 μm2 upto >2200 μm2 within Kölliker’s organ involving about 75 ISCs. Sources and directions of the waves were random, indicating that the tissue had not been impaled in or near the field of view and that ATP potentially leaking out from injured or dying cells at the outermost edges of the tissue piece did not affect ISCs and IHCs in the field of view.Using Fluo-8 AM-incubated explants of the apical organ of Corti, which were dissected with greatest care, we observed spontaneously occurring Ca2+ signals. Two rows of ISCs and the basolateral poles of eight IHCs are visible in the transmitted light image with the focus set at the maximum width of IHC nuclei  eliminates different raw fluorescence values  starting at t = 0 s , whereas the fCaTs in IHCs 4 and 7 started after Ca2+ had risen in the nearby ISC III. While IHC 7 produced 4 fCaTs shown at the onset of the recording, no elevation in Ca2+ could be observed in ISCs  and decayed to baseline after 895.9 ± 381.2 ms . Decay time defined as time from peak back to 10% above baseline was 879.7 ± 379.1 ms (n = 17), thus revealing a much slower decay in fluorescence compared to the rise , which required a smaller scan field not allowing for simultaneous imaging of ISCs. During a 573 s-long recording of eight IHCs, seven of them generated fCaTs. Single fCaTs, which were not generated within a burst of activity, had an average half-width of 32.5 ± 9.8 ms  whereas minibursts contained an average number of 2.8 ± 1.0 fCaTs  perfusion was switched off in a set of experiments to exclude the possibility that the bath flow may have caused an interaction between ISCs and IHCs. Fifty IHCs from four control mice were monitored in seven recordings lasting between 544 s and 1120 s. The incidence of fCaTs was analyzed during a total observation time of 598 min , irrespective of Cas Figure . A detai2+ dependance of fCaTs, nominally Ca2+-free solution was applied. To achieve perfusion of the cell bodies under the reticular lamina, the application pipette here was positioned at a lateral edge of the epithelium, in contrast to the setting shown in Figure 2+-free solution for >1 min, single fCaTs, minibursts and bursts were reversibly abolished  extracellular Ca2+ concentration  temporally matching the Ca2+ signals of the ISC  Ca2+ influx through the small percentage of Ca2+ channels other than Cav1.3  triggered bursts in IHCs 1 and 2, and although ISC IV was silent, IHCs 3–5 were active at the same time. The Ca2+ elevation of ISC I preceded that of the IHCs . Though less frequent, synchronized IHC bursts were also observed without any ISC activity within the recording frame as e.g., in IHCs 1, 2, 4, 5, 6, 8 at t ~90 s , spiral ganglion tissue and IHCs), indicating an overall expression of purinergic receptors at the age of P4/P5. The ATP-evoked increase in ΔF/F0 in ISCs resembled that induced by a spontaneous Ca2+ wave, lasting for 10–15 s  did not deplete the burst responses in IHCs . When taking into account that the solution flow reached the IHCs ~20 ms earlier than the ISCs, these results still indicate that ATP can elicit bursts in IHCs independent of ISC Ca2+ elevations.To validate previous results showing ATP-induced inward currents in neonatal IHCs  to 1.0 waves/100 s . After removal of PPADS, the ability of ISCs to generate Ca2+ waves was very weak, as only three Ca2+ waves occurred during 1910 s washout  and P2X7R knockout (P2X7R−/−) mice. Both, ISC waves and IHC fCaTs were unaffected in P2X2R−/−/P2X3R−/− mice . Examples of IHC Ca2+ signals from P2X2R−/−/P2X3R−/− mice are shown in Figure −/− mice involving 48 IHCs  revealed similar IHCs fCaTs and ISC Ca2+ waves as in control mice  ATP strongly promoted generation of IHC bursts Figure ; (ii) IH Figures . As in c Figures . Overall Figures . Burst-l Figures . Of the 2+ APs independently of Ca2+ wave activity in adjacent ISCs . However, not every ISC Ca2+ wave elicited burst-like activity in the adjacent IHC 2 . The relative incidence of synchronous miniburst activity in at least two adjacent IHCs without being triggered by an ISC Ca2+ was 40.3% (149 cases) compared with 59.7% (221 cases) with a Ca2+ wave as trigger . Class 2 denotes a small ISC Ca2+ wave that radially propagated ≤2 cells from its origin , whereas class 3 defines a large ISC Ca2+ wave propagating >2 cells from its origin  or showed class 1 events . However, bursts predominated when IHCs were activated by class 3 waves. When ISCs were silent (class 0), the incidence of inactive IHCs was 57% ± 1.8% (n = 196) and most of the autonomous activity observed were single fCaTs (22% ± 1.4%) or minibursts (18% ± 1.3%). During a non-propagating Ca2+ transient of a single ISC (class 1), a similar picture emerged as with silent ISCs  the incidence of bursts increased to 17% ± 6.8% and of minibursts to 23% ± 4.5%, whereas the proportion of inactive IHCs was reduced to 40% ± 10.8%. In the presence of class 3 waves (n = 19), the fraction of bursts increased to 64% ± 7% and that of minibursts to 25% ± 5.7%. In contrast, the incidence of single fCaTs or inactive IHCs was reduced to 5% ± 2.8% and 5% ± 2%, respectively. These data indicate that most bursts of fCaTs reflecting burst-like action potentials of IHCs require ISC Ca2+ waves. On the other hand, IHCs can generate single fCaTs and minibursts without wave activity of ISCs.Relating the class of ISC activity to the highest rank of the simultaneous IHC activity revealed striking differences Figure : bursts s Figure . However2+ signals in IHC and their dependance on ISC Ca2+ waves was presently studied with Fluo-8 AM Ca2+ imaging on freshly dissected explants of organs of Corti. Since ATP can trigger Ca2+ waves in Kölliker’s organ of the immature organ of Corti  and bursts of fCaTs. Our ΔF/F0 values of fCaTs in Fluo-8 AM-loaded IHCs varied from 0.5 up to 5 (in bursts), providing a much higher amplitude resolution than previously reported using Fluo-4 AM  AP repolarization by delayed rectifier K+ and SK2 channels . It is rather likely that sizes and properties of IHC currents, such as Cav1.3 currents, various K+ currents including SK2, transducer currents at rest, purinergic currents, cholinergic currents  in the absence of ISC Ca2+ waves  and its limited duration suggest that a rising Ca2+ imaging data. TE, KB and IM analyzed the data. TE, KB, IM and JE prepared the manuscript.JE and TE designed the study. TE and KB acquired CaThe 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 reviewer RL and handling Editor declared their shared affiliation at the time of review.


Ca2+ imaging revealed that TTX, L-NNA and ODQ increased Ca2+ transient firing in colonic ICC. Our results suggest that tonic inhibition in the proximal colon occurs through suppression of Ca2+ release events in ICC. Suppression of Ca2+ release in ICC limits the open probability of Ano1 channels, reducing the excitability of electrically-coupled SMCs.Spontaneous excitability and contractions of colonic smooth muscle cells (SMCs) are normally suppressed by inputs from inhibitory motor neurons, a behavior known as tonic inhibition. The post-junctional cell(s) mediating tonic inhibition have not been elucidated. We investigated the post-junctional cells mediating tonic inhibition in the proximal colon and whether tonic inhibition results from suppression of the activity of Ano1 channels, which are expressed exclusively in interstitial cells of Cajal (ICC). We found that tetrodotoxin (TTX), an inhibitor of nitric oxide (NO) synthesis, L-NNA, and an inhibitor of soluble guanylyl cyclase, ODQ, greatly enhanced colonic contractions. Ano1 antagonists, benzbromarone and Ani9 inhibited the effects of TTX, L-NNA and ODQ. Ano1 channels are activated by Ca Together these cells make up a complex of electrically-coupled cells, known collectively as the SIP syncytium2.Contractions of the smooth muscle cells (SMCs) in the proximal colon are essential for colonic motility that assists in reabsorption of water and electrolytes and eventually propels fecal materials toward the distal colon and rectum. Contractions of the proximal colon are regulated by intrinsic and extrinsic motor neurons, but neural controls are superimposed upon myogenic mechanisms that set the excitability of SMCs. The term ‘myogenic’, once exclusive to the cellular mechanisms of SMCs, now includes mechanisms attributed to interstitial cells, such as interstitial cells of Cajal (ICC) and platelet-derived-growth-factor-receptor-alpha2+ entry and release events that activate Ca2+-activated Cl− channels encoded by Ano16. Ano1 mediates inward currents, and activation or suppression of these events, affects the basal excitability of SMCs. Activation of Ano1 channels is responsible for the pacemaker events that generate propagating electrical slow waves, and these channels are also involved in responses to some enteric motor neurotransmitters8. In contrast, PDGFRα+ cells regulate colonic motility by exerting a hyperpolarizing effect on the SIP syncytium through the activation of small conductance Ca2+-activated K+ (SK) channels13.ICC regulate gastrointestinal (GI) motility through Ca2+ channels. If left alone SMCs generate action potentials, but action potentials are unable to propagate over great distances in smooth muscle syncytia and entrain the behaviors of other cells14. Therefore, the intrinsic excitability of SMCs on its own generates localized excitation and fibrillatory- like contractile behavior that is non-productive for propulsion of luminal contents. To generate orderly and propulsive contractions, the activity of SMCs must be suppressed so inputs from ICC and motor neurons can coordinate contractions. Suppression of SMC excitability is known as ‘tonic inhibition’, a major form of motility behavior in muscles of the distal GI tract and recognized for decades15. Tonic inhibition in colonic muscles results from release of inhibitory neurotransmitters. Tetrodotoxin (TTX) or nitric oxide (NO) synthase inhibitors, such as Nω-Nitro-L-arginine (L-NNA) increase the excitability of colonic muscles from mouse18, rat22, cat23, canine24 and human26. Tonic inhibition leads to relative electrical and mechanical quiescence between colonic migrating motor complexes (CMMCs), and muscle excitability is thought to increase due to suppression of inhibitory neurons and firing of excitatory neurons at the onset of the CMMC27. Impairment of tonic inhibition has been linked to conditions such as chronic constipation and the pseudo-obstruction that occurs in infants with Hirschprungs disease28. However, the post-junctional cell(s) and intracellular mechanisms responsible for linking neural input to tonic inhibition are not well understood.Colonic SMCs are intrinsically active spontaneously due to their resting potentials that are at or near the threshold for activation of voltage-dependent Ca2+-activated Cl− channel encoded by Ano129. Ca2+ transients in ICC, due to release from intracellular Ca2+ stores, are coupled to activation of Ano130 and we found recently that NO suppresses Ca2+ transients in ICC from the small intestine32. Ano1 channels are expressed exclusively in ICC in GI muscles29. Therefore, we investigated whether tonic inhibition, due to nitrergic mechanisms in the proximal colon, occurs as a result of mechanisms present in ICC. Results are presented showing that nitrergic inputs suppress Ca2+ release events in ICC in the proximal colon, and the enhancement in contractions elicited by blocking tonic inhibition are suppressed by Ano1 channel antagonists.ICC in the colon generate spontaneous transient inward currents (STICs) due to expression of CaMurine proximal colon muscles exhibited spontaneous phasic contractions Fig. . Under b33. We tested an inhibitor of sGC, 1H- Oxadiazolo  quinoxalin-1-one (ODQ), to determine whether blocking this signaling pathway had the same effects as TTX and L-NNA. ODQ (10 μM) increased AUC 3.2 ± 0.2 fold  antagonist  did not cause an appreciable increase in proximal colon contractions  and generation of cyclic guanosine monophosphate (cGMP)36 benzbromarone  used in the study of Ano1 antagonists. Long-term application (up to 60 min) of DMSO (0.03%) to muscles after TTX had no significant effects on colonic contractions  stores via Ca2+ channels such as inositol-tri-phosphate receptors (IP3Rs) and ryanodine receptors (RyRs). Sustained Ca2+ release from stores requires refilling by the sarco/endoplasmic-reticulum-Ca2+-ATPase (SERCA) pump37. Therefore, we tested the effects of the SERCA pump inhibitor thapsigargin (1–10 μM) on the excitatory effects of TTX, L-NNA and ODQ. Thapsigargin reduced the excitatory effects induced by TTX  was found previously to be important in ICC for sustaining Ca2+ release events and maintaining pacemaker activity in the GI muscles38. In ICC, SOCE appears to be mediated by interactions between the ER protein, stromal interacting molecule (STIM), and Orai, a family of Ca2+ influx channels in the plasma membrane38. We tested the effects of an Orai channel blocker (GSK 7975 A) on the contractile activity enhanced by TTX, L-NNA and ODQ. GSK 7975 A inhibited the contractions after TTX  reduced the AUC of colonic contractions in the presence of TTX from 2.6 ± 0.3 to 1.7 ± 0.3 . Intramuscular ICC (ICC-IM) were imaged in these experiments, as these cells are thought to be involved in neuro-effector transduction in GI muscles40. Colonic ICC-IM generated spontaneous Ca2+ transients similar to those previously characterized in the ICC in the region of the deep muscular plexus in the small intestine31. Spatio-temporal maps (STMs) of Ca2+ transients recorded from ICC-IM in the proximal colon in situ revealed that Ca2+ transients arose from multiple sites along the lengths of individual ICC-IM and were stochastic in their firing patterns  on contractile responses of proximal colon muscles to elevated external K+ ([K+]o). These experiments were performed in the presence of TTX (1 μM), L-NNA (100 μM) and atropine (1 μM) to minimize confounding effects from depolarization-dependent release of major motor neurotransmitters. Elevated [K+]o (60 mM) evoked reproducible contractures in colon muscle. Benzbromarone (1 μM) reduced elevated [K+]o contractions to 56.4 ± 3.7% of control , inhibition of nNOS (L-NNA) or blocking the receptor for NO (ODQ) unleashed increases in myogenic contractions of the mouse proximal colon. Localized Ca2+ transients were observed in ICC-IM of the proximal colon that have been attributed to release of Ca2+ from stores and coupled to activation of Ano1 Cl− channels in other classes of ICC32. The agents that block the nitrergic pathway greatly increased the Ca2+ transients in ICC-IM and increased the contractile responses of intact muscles. Ano1 channel antagonists, thapsigargin and the Orai channel blocking drug, GSK7975A, inhibited this contractile activity. Taken together, these data suggest that tonic inhibition  results from suppression of Ca2+ release events in ICC, which reduces the activation of Ano1 channels and maintains a low level of excitability in the SIP syncytium. Another interpretation of our results could be that the Ano1 antagonists block voltage-dependent Ca2+ channels, and this is the cause of colonic inhibition. While control experiments showed this is a concern with benzbromarone, as discussed below, significant effects on colonic contractions were observed with Ani9 at a concentration that blocks Ano1 channels35 and had little or no effect on elevated [K+]o induced contractions.Tonic inhibition is a well-known motor phenomenon in GI muscles of mice42. Transduction of nitrergic inputs mediated by ICC most likely results in electrical responses (e.g. hyperpolarization due to openings of K+ channels or reduced activation of inward currents due to Cl− or non-selective channels)42 that are conducted to electrically coupled SMCs in the SIP syncytium. Evidence for innervation and transduction of NO by ICC includes: (i) very close associations (<20 nm) between ICC and nitrergic nerve terminals44, (ii) neural inhibition of spontaneous Ca2+ transients in ICC that can be relieved by TTX or L-NNA46, (iii) expression of signaling molecules required for post-junctional nitrergic responses in ICC43; (iv) reduction or loss of nitrergic responses in animals with ICC-specific knockdown of nitrergic signaling molecules49.NO released from enteric inhibitory neurons is thought to be transduced, in part, by ICC Fig. 42. Trans29, and activation of these channels is required for the principal functions of ICC, pacemaking and neurotransduction, because genetic ablation of Ano1 causes loss of pacemaker activity in the stomach and small intestine of juvenile animals50 and reduced post junctional responses to nerve stimulation in the stomach51. Activation of Ano1 in ICC is due to Ca2+ release from intracellular ER stores6, and Ca2+ release and the spontaneous transient inward currents (STICs) resulting from activation of Ano1 channels are inhibited by blocking the refilling of the ER with the SERCA pump inhibitor, thapsigargin31, or by depleting ER stores by inhibiting SOCE in ICC38. We reasoned that Ano1 antagonists are a useful tool for determining the role of Ano1 channels, and thus mechanisms intrinsic to ICC, in GI motor patterns. Since Ano1 channels in ICC are activated by Ca2+ release we also reasoned that interfering with Ca2+ release by inhibiting the SERCA pump or SOCE may also indicate an ICC-dependent mechanism, as excitation-contraction coupling in SMCs is primarily dependent upon Ca2+ entry via voltage-dependent Ca2+ channels and not Ca2+ release mechanisms52. We found that Ca2+ handling antagonists, thapsigargin and the Orai channel inhibitor central to SOCE, GSK 7975 A, significantly reduced the colonic contractions unleashed by TTX, L-NNA and ODQ. These observations are consistent with the idea that NO-dependent inhibition of Ca2+ release in ICC reduces activation of Ano1 and this causes reduced SMC excitability and contraction.Ano1 channels are expressed exclusively in ICC in GI muscles2+ handling in ICC is poorly understood. Certain phosphorylation-dependent mechanisms can modulate the activation of ER Ca2+ release channels, such as IP3Rs53. Either cGMP-dependent protein kinase G (PKG)55 phosphorylation of IP3R or a downstream modulator of NO signaling, such as IP3R-associated cGMP-kinase substrate 56 might affect Ca2+ release in response to nitrergic stimulation. PKG, as well as protein kinase A, have been shown to limit Ca2+ release in myocytes and endothelial cells of the vasculature57 and in interstitial cells from the rabbit urethra58. IRAG has been shown to be involved in cGMP-dependent regulation of Ca2+ release in model cells56 and in NO-mediated relaxation of human colon59. IP3Rs are known to facilitate Ca2+ release in ICC31, thus its regulation would be expected to modulate Ca2+ transients in ICC. More in depth studies of the downstream coupling between NO-dependent cGMP production and Ca2+ release in ICC should be a goal of future studies.At present, how NO interferes with Ca+ cells express P2Y1 receptors and SK3 channels in abundance and appear to mediate purinergic responses in the colon through P2Y1 receptor pathways60. These cells also express signaling molecules, such as the subunits of guanylate cyclase and PKG61. PDGFRα+ cells via purinergic inhibition are not likely to be involved in tonic inhibition in the proximal colon, because a P2Y1 receptor antagonist, MRS 2500, did not increase contractions. The question of mediation of nitrergic tonic inhibition is more difficult, but peripheral information suggests that PDGFRα+ cells are unlikely to contribute to nitrergic inhibition in the colon. NO causes inhibition of Ca2+ release in ICC  in either SMCs or ICC suggested that both ICC and SMCs are responsible for the effects of NO on contractile frequency in the proximal colon62. The mechanism for increasing contractile frequency in the sGC-SMC knock-down was unexplained, but a possible mechanism for increasing the force of contractions in SMCs directly might be changes in Ca2+ sensitivity of the contractile apparatus. Ca2+ sensitivity in SMCs is regulated by cyclic nucleotides, such that cGMP-dependent mechanisms would tend to reduce the force of contractions for a given level of Ca2+ in SMCs. In general, the response to Ano1 antagonists in our study, an effect on channels in ICC, was a reduction in contractile frequency. One would expect that effects on Ca2+ sensitivity would be changes in contractile amplitude, a response intrinsic to SMCs, without effect on contractile frequency.While our data indicate that ICC bear significant responsibility for mediation of tonic inhibition, other cells that are part of the SIP syncytium must be considered for contributions. PDGFRαICC Fig. , but if 65, and Ani9 (1 μM), a newer Ano1 antagonist, was shown to block currents due to expression of human ANO1(abc splice variant) expressed in Fisher rat thyroid (FRT) cells (i.e. IC50 of ~110 nM)35. However, the specter of off-target effects is always of concern with antagonists of Ca2+-activated Cl− channels. In fact several Ano1 antagonists, said to be selective, have been shown to inhibit L-type Ca2+ channels. Such an effect could provide an alternative explanation to our findings because colonic contractions might also be inhibited by blocking L-type Ca2+ channels and reducing Ca2+ influx into SMCs  also reduced KCl contractions by ~30%  through Ano1 channels  that suppresses Ano1 current nearly 100%35. The results of this study demonstrate a novel function of ICC in regulating the motility pattern known as tonic inhibition in the colon.In summary, tonic inhibition, a well-described and important motility mechanism that suppresses the excitability and contractile activity of colonic SMCs, is mediated in part by ICC by suppression of Caels Fig. . SuppresAnimals used and the protocols performed in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno.2 2.5, MgCl2 1.2, NaHCO3 23.8, KH2PO4 1.2, dextrose 11.0. This solution had a pH of 7.4 at 37 °C when bubbled to equilibrium with 95% O2–5% CO2. Colons were removed from mice, pinned down in a Sylgard-lined dish and opened longitudinally. Mucosa and submucosa were peeled away leaving the tunica muscularis intact.For contractile studies, wildtype mice  were anaesthetized by isoflurane inhalation  and then sacrificed by cervical dislocation. The GI tract en masse was removed through an abdominal incision and transferred to a dish containing Krebs-Ringer-bicarbonate (KRB) solution of the following composition (in mM): NaCl 118.5, KCl 4.7, CaCl2+ imaging experiments, we utilized a genetically-encoded Ca2+ indicator, GCaMP6f, expressed exclusively in ICC using the Cre-Lox P technique, where an inducible Cre Recombinase (induced by tamoxifen injection) was driven by an ICC specific promoter (tyrosine kinase receptor Kit). Ai95 (RCL-GCaMP6f)-D (GCaMP6f mice) were purchased from the Jackson Laboratory . c-Kit+/Cre-ERT2 (Kit-Cre mice) were gifted from Dr. Dieter Saur of the Technical University Munich, Germany. GCaMP6f mice were crossed with Kit-Cre mice and the resultant offspring are referred to as Kit-Cre-GCaMP6f mice throughout the manuscript. These animals were injected with tamoxifen at ages of 6–8 weeks to induce Cre Recombinase and subsequent GCaMP6f expression. Tamoxifen  was dissolved in 800 μL of ethanol  by vigorous vortexing for 20 minutes. Then, 3.2 ml of Safflower (generic) was added to create solutions of 20 mg/ml, which were then sonicated for 30 minutes prior to injection. Mice were injected  with 0.1 ml of tamoxifen solution (2 mg tamoxifen) for three consecutive days. Mice were used 10 days after the first injection and the subsequent expression of GCaMP6f was confirmed by genotyping. Kit-Cre-GCaMP6f mice were sacrificed and flat sheets of proximal colon, with the mucosa removed were prepared in the same manner as wildtype animals.For ICC CaMuscle strips (5–8 mm in length and ~2 mm in diameter) were cut parallel to the circular muscle fibers from the proximal colon, attached to a stable mount and to a Gould strain gauge and immersed in jacketed 15 ml tissue baths. The muscles were continuously oxygenated and maintained in KRB solution at 37 °C. Tissues were stretched to an initial tension of 5 mN, and experiments began after 60 mins of equilibration. Tissues were washed with fresh, oxygenated KRB solution every 20 mins. Isometric contractions were recorded using AcqKnowledge software .2+ transients in ICC in situ was acquired using an Eclipse E600FN microscope  equipped with a 60 × 1.0 CFI Fluor lens . GCaMP6f was excited at 488 nm , as previously described66. The pixel size using this acquisition configuration was 0.225 µm. Image sequences were collected at 33 fps with TILLvisION software . Ca2+ imaging experiments were performed in the presence of nicardipine (100 nM) to minimize movement artefacts resulting from contractions. For experiments with pharmacological interventions, a control period of 30 sec was recorded and then KRB solution containing the appropriate concentration of drug was perfused into the bath for 15 mins to allow for full tissue penetration before acquiring another 30 sec recording to ascertain the effects of the drug on ICC.The proximal colon was pinned with the circular muscle (CM) layer facing upward to the base of a 5 ml chamber (60 mm diameter) coated with Sylgard. The muscles were continuously perfused with warmed KRB solution at 37 °C for an equilibration period of 1 hour before experimentation. Following this equilibration period, imaging of Ca2+ transients were quantified with spatio-temporal maps as described previously67. Briefly, movies of Ca2+ activity in ICC were converted to a stack of TIFF (tagged image file format) images and spatio-temporal maps (STMs) of Ca2+ activity were generated in individual ICC within a FOV using Image J . The basal Ca2+ fluorescence was acquired from regions of the cell displaying the most uniform and least intense fluorescence (F0), the fluorescence value throughout the cell was then divided by the F0 value to calibrate the STM for amplitude of Ca2+ events as F/F0. Ca2+ transient frequency in ICC was expressed as the number of events fired per minute (min−1). The amplitude of Ca2+ transients was expressed as ΔF/F0, the duration of Ca2+ transients was expressed as full duration at half maximum amplitude (FDHM) and the spatial spread of Ca2+ transients was expressed as μm of cell propagated per Ca2+ transient. In experiments where drugs were applied to ICC activity, quantification of Ca2+ transient parameters in control and in the presence of a drug would be analyzed in 3–5 paired representative cells in a FOV per animal used in experimentation.Isometric contractions were analyzed using Clampfit software (Axon Instruments). Area under the curve (AUC) measurements from control periods were normalized to 1.0, and changes in AUC were normalized as a fold changes from the control values. AUC under control conditions was tabulated during the 10 min immediately before addition of the drugs, and responses to the drugs were tabulated from the period 10–20 min after adding the drug. For imaging files, CaThroughout the manuscript, n values represent the number of muscle strips or cells in a dataset, and all datasets represent data from a minimum of 3 animals. Data were analyzed for statistical significance using Prism , and values are expressed as mean ± SEM. Statistical analyses used a paired students t test or a two way ANOVA with a post hoc Tukey multiple comparison test as appropriate. P values < 0.05 were considered to be statistically significant.H- Oxadiazolo  quinoxalin-1-one (ODQ), and Thapsigargin were purchased from Tocris Bioscience . GSK 7975 A was purchased from Aobious .Tetrodotoxin (TTX), 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl) methylideneamino]-acetamide (Ani-9), Benzbromarone, Nω-nitro-L-arginine (L-NNA), 1Supplementary Information


Acidity at surface of cancer cells is a hallmark of tumor microenvironments, which does not depend on tumor perfusion, thus it may serve as a general biomarker for targeting tumor cells. We used the pH (low) insertion peptide (pHLIP) for decoration of liposomes and niosomes. pHLIP senses pH at the surface of cancer cells and inserts into the membrane of targeted cells, and brings nanomaterial to close proximity of cellular membrane. DMPC liposomes and Tween 20 or Span 20 niosomes with and without pHLIP in their coating were fully characterized in order to obtain fundamental understanding on nanocarrier features and facilitate the rational design of acidity sensitive nanovectors. The samples stability over time and in presence of serum was demonstrated. The size, ζ-potential, and morphology of nanovectors, as well as their ability to entrap a hydrophilic probe and modulate its release were investigated. pHLIP decorated vesicles could be useful to obtain a prolonged (modified) release of biological active substances for targeting tumors and other acidic diseased tissues.The online version of this article (10.1186/s11671-018-2807-8) contains supplementary material, which is available to authorized users. About a century ago, Paul Ehrlich floated the idea of realizing “magic bullets” to specific and more effective delivery of drugs . The “maIn 1965, Bangham observed phospholipid-based vesicles for the first time  and, in Liposome size is a critical parameter in influencing the carrier fate after administration, in terms of plasma proteins absorption, recognition by reticuloendothelial system (RES), circulation half time, and cellular trafficking. Nanosized carriers can favor cell internalization and tumor targeting, thus many researchers have been focusing on “nanonization” –9.non-ionic liposomes, niosomes, or non-ionic surfactant vesicles). Sorbitan esters surfactants (Spans®) are lipophilic substances widely used in niosome preparation. In order to prolong the vesicles circulation time and obtain “stealth” nanocarriers, polyethylene glycol (PEG) incorporation is a gold standard approach: through this conjugation, ethoxyethylatedsorbitan esters surfactants (Tweens®) are obtained. Both Span and Tween are characterized by a different hydrophilic/lipophilic balance (HLB) value and the choice of the surfactant allows preparing niosomes with the desired properties  sodium salt  were purchased from Avanti Polar Lipids and pyrene was obtained from Fluka. pHLIP peptide (ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT) was synthesized and purified by CS Bio. All other products and reagents were of analytical grade.Polyoxyethylenesorbitan monolaurate (Tween 20), sorbitan monolaurate (Span 20), cholesterol (Chol), Hepes salt {ε280 = 13,940 M−1 cm−1.pHLIP was conjugated with DSPE lipids in methanol by the covalent conjugation of DSPE-maleimide with the single cysteine residue at the N-terminus of pHLIP, as it was described previously , 21, 22.The thin layer evaporation method was used to prepare non-ionic surfactant (from Tween 20 or Span 20) and phospholipid (from DMPC) vesicles, with and without pHLIP. In each vesicle formulation, cholesterol was added in different molar ratios Table  23].Tab.Tab23].TSample composition has been optimized choosing previously well-characterized structures , 25 at w3:CH3OH mixture (3:1); the organic solvent was then removed under vacuum at different temperatures depending on the sample. The obtained film was hydrated with 5 mL of Hepes buffer (0.01 M pH 7.4) or sodium calcein solution 10−2 M. The suspension was vortex-mixed for about 5 min, followed by sonication  and Hepes buffer as eluent. Afterwards, the purified vesicle suspension was filtered by means of cellulose filters with the appropriate pore diameter.The same preparation method was used to prepare pHLIP-coated niosomes and liposomes.T = 25 °C by dynamic light scattering (DLS), using a Malvern NanoZetaSizer ZS90, equipped with a 5 mW HeNe laser (wavelength λ = 632.8 nm) and a digital correlator. The normalized autocorrelation functions of the scattered intensity at 90° angle were analyzed by the Contin algorithm to obtain the distribution of the particles diffusion coefficient D, hence the distribution of the effective hydrodynamic radius RH of the vesicles via the Stokes-Einstein relation RH = KBT/6πηD, where KBT is the thermal energy and η is the solvent viscosity. The width of the size distribution of niosomes/liposomes is rather small but non-negligible. The values reported in Table The average size and size distribution of the vesicles were measured at u was converted into the ζ-potential using the Smoluchowski relation ζ = uη/є, where η and є are the viscosity and the permittivity of the solvent phase, respectively [The electrophoretic mobility measurements were carried out by the laser Doppler electrophoresis technique, using the Malvern NanoZetaSizer ZS90 apparatus. The mobility ectively .−1 ≤ q ≤ 6 nm−1, q = (4π/λ)sin(θ/2), where θ is the scattering angle and λ = 0.1 nm the X-ray wavelength. The corresponding investigated length-scale is between 1 and 60 nm, suitable for accessing the internal structure of niosomal and liposomal vesicles. All experiments were performed at T = 25 °C with short irradiation time, 0.1 s, to prevent radiation damage. For each sample, the scattered intensity at different scattering angles was captured on a 2D detector, then angularly regrouped, subtracted for the background and solvent contributions, and analyzed to obtain information on the shape and internal structure of the vesicles in solution.Small angle X-ray scattering (SAXS) experiments were carried out at European Synchrotron Radiation Facility . The use of the ID02 high-brilliance beamline allowed to perform measures on dilute solutions in the region of momentum transfer 0.1 nmIn the case of unilamellar vesicles, the closed bilayer was modeled with three concentric shells corresponding to the external headgroups, the hydrophobic chains, and the internal headgroups. A Schulz distribution for the vesicle size was assumed.Vesicle solution (5 μL droplet) was spread on a Lacey formar/carbon electron microscopy grid and preserved in a frozen-hydrated state by a rapid freezing in liquid ethane. The vitrification process was performed using FEI Vitrobot system with the setting of a single blot of 3 s, an offset of 1, and drain and waiting time of 1 s. Transmission electron microscopy (TEM) (JEOL 2100) with an accelerating voltage of 200 kV at magnifications in the range of × 10,000 to × 150,000 was used to image vesicles.Physical stability studies of niosomes and liposomes, prepared with and without pHLIP, were carried out at two different storage temperatures (25 °C and 4 °C). The aim was to evaluate if significant changes in size and ζ-potential of the vesicles dispersion occur over a period of 90 days. Samples from each batch were withdrawn at definite time intervals  and the vesicles size and ζ-potential were determined as previously described.Similarly, the vesicles stability was investigated also in the presence of biological fluids, such as human serum. The suspensions were contacted with 45% of human serum at 37 °C. At definite time intervals , variations in vesicle size and ζ-potential were determined as described.Fluidity and microviscosity-polarity of niosomal and liposomal bilayers were accessed by fluorescence measurements of two fluorescent probes, diphenylhexatriene (DPH) and pyrene, respectively, located in the hydrophobic region of the bilayer membrane. Both DPH (2 mM) and pyrene (4 mM), 220 μL and 2.5 mg respectively, were added to the surfactant or phospholipid/cholesterol mixture before vesicle preparation . Afterwaexc = 400 nm and λem = 425 nm and the fluorescence anisotropy (A) of samples was calculated according to the following equation [The fluorescence anisotropy measurements were carried out at room temperature using an LS55 spectrofluorometer  at λequation :Ivv and Ivh are the intensities of the emitted fluorescence (arbitrary units), respectively, parallel and perpendicular to the direction of the vertically polarized excitation light. The correction factor G = Ihv/Ihh is the ratio between the vertically and the horizontally polarized emission components when the excitation light is horizontally polarized. The fluorescence anisotropy values are inversely proportional to membrane fluidity. Therefore, high fluorescence anisotropy value corresponds to a high structural order and/or low membrane fluidity [where fluidity .exc = 330 nm, recording the emission spectrum in the range 350 < λem < 550 nm [I1/I3, is related to the polarity of the pyrene environment [I1/I3 ratio correspond to a nonpolar environment. Further, pyrene molecules incorporated into the vesicle bilayer may form intramolecular excimers and this process is sensitive to the viscosity of the probe microenvironment [IE/IM, is related to microviscosity.In order to evaluate the microviscosity and the micropolarity of the vesicle bilayer, fluorescence experiments with pyrene were carried out with a Perkin-Elmer LS55 spectrofluorometer at λ< 550 nm . The finironment . Indeed,ironment . TherefoThese studies were carried out on the vesicles prepared both with and without pHLIP and the obtained results were then compared.−2 M. At this concentration, the calcein fluorescence is self-quenched [Non-ionic surfactant or phospholipid vesicles were loaded with calcein at concentration of 10quenched . Dequencquenched .ex = 492 and λem = 520 nm. The reference value F∞ (arbitrary units), correlated to the total calcein amount entrapped in the vesicles [v/v). The release percentages (F%) at each time point were obtained using the following equation:The calcein release was monitored by measurements of the fluorescence of the medium using a Perkin-Elmer LS50B spectrofluorometer with λvesicles , was detFt (arbitrary units) is the fluorescence read at a specific time point t.where t test was carried out to evaluate the statistical significance of the ANOVA test. A p value < 0.05 was considered statistically significant. Results are the average of three experiments ± standard deviation.A one-way ANOVA was used for statistical analysis. A posteriori Bonferroni Tween20-, Span20-, and DMPC-vesicles, prepared with and without DSPE-pHLIP, were characterized for size and ζ-potential . Span20-based vesicles are definitely multilamellar, with a characteristic peak at q = 1.6 nm−1 corresponding to an interlamellar distance of 3.8 nm, matching twice the length of the Span20 molecule. Results indicate that the multilayered shell of Span20 vesicles is composed of adjacent bilayers with almost no interlamellar water.Tween20-based niosomes are unilamellar, while DMPC vesicles display a certain degree of multilamellarity (as liposomes), as shown by the presence of the characteristic peak at q-range is quite similar, indicating comparable fraction of structured material and comparable overall size of the vesicular particles.The presence of DSPE-pHLIP (less than 1% mole fraction) does not dramatically affect the systems. The scattered intensity in all the investigated q = 1 nm−1. The insertion of a low fraction of DSPE C18-chains, linked to each peptide molecule, does not change the thickness of the lipid bilayer, and mainly dictated by the large fraction of cholesterol with respect to DMPC (about 40–50%). At this concentration of cholesterol, lipid chains are organized in the liquid ordered (Lo) phase, and characterized by the decoupling of the weak lateral positional order of the chains with respect to the high orientational order along the chains and capable of hosting few C18-chains without any structural change [DMPC-based vesicles show a slight increase in the number of bilayers within the liposome, as deducible by the intensity increase of the Bragg peak at l change . Also Spq = 1.84 nm−1 (characteristic distance of 3.41 nm), and the vesicle structure is unaffected. The presence of few microcrystallites has been often found associated with Tween-based niosomes [3), together with an increase in the roughness of the layer. This result indicates that addition of DSPE-pHLIP mainly affects the external shell of the vesicles. This is consistent with a picture where the DSPE C18-chains insert into the bilayer and anchor the peptide to the vesicle surface, among the extended and ramified polyethylene-glycol headgroups. We note that the ability of pHLIP peptide itself (not linked to the lipid molecules) to interact with the external surface of vesicles at high pH and insert into bilayer at low pH has been recently observed by SAXS in the case of unsaturated phospholipid bilayers [In the Tween20 + pHLIP system, a small amount of cholesterol crystallites is present, as shown by the typical sharp peak at niosomes  in amounbilayers . In the To investigate whether the pHLIP presence could affect bilayer microrheological properties , a lipophilic shell characterization was carried out. The characterization studies were conducted by using the fluorescent probe pyrene, added to the samples at the beginning of the preparation procedure. Due to its lipophilic nature, it inserts into the vesicle bilayer and provides information about the polarity and microviscosity of the membrane environment.As shown in Table The bilayer was further characterized by measurements of fluorescence anisotropy of another lipophilic fluorescent probe, DPH, incorporated into lipids. These measurements reflect the probe movement and its orientation within the bilayer which gives information on the bilayer fluidity that can affect the vesicles capability to release their content . FluoresThe anisotropy values for each sample are shown in Table Further characterization studies were conducted on the examined preparations. All the samples, prepared with and without pHLIP, were stored for 90 days at 4 °C temperature in order to evaluate stability of the vesicles over time, by measuring their size and ζ-potential variations  human serum and stored at 37 °C for 3 h . This event can be due to the absorption of plasma proteins on the niosomal surface, as result of electrostatic interactions, however, without incurring in vesicles disruption. This hypothesis is supported by the ζ-potential decrease after the vesicles contact with human serum. This phenomenon occurs when the ζ-potential reaches slightly negative values, around the neutrality region, making the system to be unstable, and vesicles start aggregating.As well as Tween20, DMPC vesicles show an increase in size in comparison to the pre-contact sample. However, this variation turned out to be non-significant and the size values maintained under 280 nm (DMPC) or under 240 nm (DMPC + pHLIP).The release capability was evaluated for the samples prepared with and without pHLIP following the amount of calcein (hydrophilic probe) released from the vesicles over the time.The studies were carried out at the temperature of 37 °C for a time period of 24 h, by exposing of the purified samples with either HEPES buffer or human serum. As shown in the Fig. The released amount of calcein was in between 30 and 50% indicating no differences in the release capabilities between the samples prepared with and without pHLIP.Same results have been obtained for Span and DMPC samples (data not shown).Comparable release profiles have been reported for different stimuli-responsive nanocarriers, such as thermo-sensitive cubosomes  or polymsn-glycero-3-phosphocholine (POPC) and does not induce fusion or membrane leakage.In conclusion, the physico-chemical differences observed in terms of microviscosity and fluidity for the samples prepared with and without pHLIP Table  do not aThis study confirms the possibility of preparing pHLIP decorated vesicles. Samples are stable if stored at 4 °C temperature for at least 3 months and in the presence of serum. Furthermore, proposed nanovectors are able to entrap a hydrophilic probe and to modulate its release.pHLIP vesicles were fully characterized in order to obtain fundamental understanding on nanocarrier features and facilitate the rational design of acidity sensitive nanovectors.The pHLIP association is driven by the hydrophobic interaction of the conjugated DSPE C18-chains inserting in the hydrophobic region of the nanovector. The pHLIP peptide is anchored and lies in the external headgroup region, prone to interact with target membranes at acidic pH and/or to release the nanovector content after bilayer rearrangement in low pH environment.According to these findings, proposed pHLIP decorated vesicles could be useful to obtain a prolonged (modified) release of biological active substances for targeting tumors and other acidic diseased tissues.Additional file 1:Table S1. Parameters for the preparation of several samples with and without pHLIP. Figure S1. SAXS intensity spectra of TWEEN20 based vesicles without (black dots) and with (red dots) pHLIP, together with the fitting curves obtained with a multishell model. The core has the same electron density of the solvent (water) and the three shells are constituted by the inner headgroups layer, the hydrophobic layer, the outer headgroups layer. Parameters, thickness and electron density are reported in the Table S2. Table S2. Parameters, thickness and electron density obtained by SAXS analysis. Figure S2. Samples stability in terms of size and ζ-potential variations over time. Figure S3. Z-Average variations for all samples in contact with human serum. The ζ-potential values are around -7 mV for all samples. (DOCX 635 kb)


Ligusticum jeholense has been used as the traditional medicine ‘Go-Bon’  in China and Korea. Considering the increased use of medicinal herbs to treat hypertension, in this study, we aimed to investigate the mechanisms of the vasorelaxation effect caused by L. jeholense. We tested the methanol (MeOH) extract of L. jeholense root and rhizoma for vasorelaxant effects; while using an isolated organ-chamber technique, L. jeholense extract (LJE) induced relaxation in the rat aortic rings by stimulating vascular endothelial and smooth muscle cells. LJE showed concentration-dependent relaxant effects on endothelium-intact and endothelium-denuded aortic rings pre-contracted with both phenylephrine (PE) and potassium chloride (KCl) in Krebs-Henseleit (KH) buffer. The vasorelaxant effect of LJE was partly attenuated by pre-treatment with glibenclamide or 4-aminopyridine (4-AP) as K+ channel blockers. Moreover, LJE showed concentration-dependent inhibition of vasoconstriction by Ca2+ supplementation in the aortic rings that were pre-contracted with PE or KCl in Ca2+-free KH buffer. In addition, a combination of LJE and nifedipine, pre-incubated further, decreased PE-induced contractions. The results suggested that LJE-induced vasorelaxation were related to blocking K+ channels and inhibiting entry of extracellular Ca2+ via receptor-operative Ca2+ channels (ROCCs) or voltage-dependent Ca2+ channels (VDCCs). High blood pressure, or hypertension, is a major determinant of mortality caused by cardiovascular disease, cerebrovascular disease, and stroke. In hypertension, vasoreactivity is an inevitable factor for the treatment of hypertension because it affects circulation and blood pressure in the cardiovascular system. Quality of life is important for antihypertensive therapy. However, although these drugs continue to be developed to treat hypertension, synthetic drugs have various adverse effects. The efficacy of these drugs increases in a dose-dependent manner, which leads to more adverse effects . In addiLigusticum (Umbelliferae), consisting of approximately 50 species growing in Asia, Europe, and North America, is complex and widespread  by the Kyung Hee University Institutional Animal Care and Use Committee. All rats were acclimated under standard laboratory conditions  and food and water were given 4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1; pH 7.4], and then aerated with a gas mixture of 95% O2–5% CO2 at 37 °C. After carefully removing the connective tissue and fat surrounding the aorta, the aorta was cut into 2-mm-long rings and suspended in organ chambers containing 10 mL KH buffer at 37 °C. The rings were suspended between two tungsten stirrups and one stirrup was connected to an isometric force transducer . The aortic ring segments were incubated under no tension for 30 min and left to equilibrate for 1 h at an optimal resting tension of 1.2 g. The KH buffer was refreshed every 15–20 min during the equilibration period for 90 min. The changes in tension of the aortic rings were recorded using isometric transducers connected to a data acquisition system . When necessary, the endothelium layer was removed by gently rubbing inside the lumen using a thin polyethylene stick. The presence of functional endothelium was checked by the ability of ACh (10 μM) to induce more than 80% relaxation in PE (1 μM)-contracted aorta rings. Endothelium-denudation was considered effectively removed when ACh caused less than 10% relaxation. Ca2+-free KH buffer was prepared by removing CaCl2 and adding EGTA (1 mM). The antagonist and inhibitor concentrations were selected in the same manner as described previously [Rats were anesthetized by exposure to ether, the thoracic aorta was removed and immersed in KH buffer [composition (mM): NaCl, 118.0; KCl, 4.7; MgSOeviously .t-test. All statistical analyses were performed by using SPSS (version 21.0) statistical analysis software . P values less than 0.05 were considered statistically significant.Results are expressed as mean ± standard error of mean (SEM). Statistical comparisons were made using Student’s 2) pathway, or muscarinic receptors transduction pathway; (2) K+ channels were partly related to LJE-induced vasorelaxation; and (3) LJE relaxed the aortic rings by blocking the entry of extracellular Ca2+ via ROCCs and VDCCs.In conclusion, (1) the vasorelaxant effects of LJE were not related to the direct NO pathway, NO-cGMP pathway, vascular prostacyclin (PGI


Presynaptic Ca2+ channels and SNARE proteins (SNAREs) interactions form a large signal transfer complex, which are core components for exocytosis. Ca2+ channels serve to regulate the activity of Ca2+ channels through direct binding and indirect activation of active zone proteins and SNAREs. The activation of Ca2+ channels promotes synaptic vesicle recruitment, docking, priming, fusion and neurotransmission release. Intracellular calcium increase is a key step for the initiation of vesicle fusion. Various voltage-gated calcium channel (VGCC) subtypes exert different physiological functions. Until now, it has not been clear how different subtypes of calcium channels integrally regulate the release of neurotransmitters within 200 μs of the AP arriving at the active zone of synaptic terminal. In this mini review, we provide a brief overview of the structure and physiological function of Ca2+ channel subtypes, interactions of Ca2+ channels and SNAREs in neurotransmitter release, and dynamic fine-tune Ca2+ channel activities by G proteins (Gβγ), multiple protein kinases and Ca2+ sensor (CaS) proteins.Action potential (AP) induces presynaptic membrane depolarization and subsequent opening of Ca High-voltage activated Ca2+ channels are composed of the pore-forming Cavα1 and four auxiliary subunits  is the largest and main subunit, which is composed of about 2000 amino-acid residues. The molecular weights of α2, β, γ and δ subunits are 143 kDa, 53–70 kDa, 30 kDa and 24–27 kDa, respectively. The Cavα1 contains four homologous domains . The transmembrane S5–S6 segments form a p loop, and the S1–S4 segments serve as the voltage sensor , Cav2.1–2.3  and Cav3.1–3.3  Ca2+ channels are present in the peripheral nervous system (PNS) and central nervous system (CNS). Though R-type Ca2+ channels are not the main Ca2+ channels, they are also involved in presynaptic plasticity and neurotransmitter release  have been classified into N-, P/Q-, R-, L- and T-type  blocks the N-type channels irreversibly in central nervous system (CNS) and Peripheral nervous system  and ω-Aga IVA blocks the Cav2.1 (Q-type) with a lower affinity in CNS  may lead to higher local presynaptic Ca2+ concentrations and frequently co-localized with synaptotagmin-containing vesicle clusters, whereas the N-type channel (Cav2.2) and R-type channel (Cav2.3) are only partially involved in vesicle clusters ]  that needs assistance by Munc13-1. Munc13-1-RIM heterodimer formation is a key component for fusion. Furthermore, the C2B domain of RIM can modulate Ca2+ channel activation  the trigger-release and spontaneous release  and transmembrane domain. Syt-1, Syt-2 and Syt-9 bind to Ca2+ to promote synchronous transmitter release, while Syt-7 evokes a asynchronous transmitter release  is a Ca2+ binding to syt abolishes the complexin clamp and triggers synaptic vesicle fusion. Recent study has revealed that complexin may regulate fusion in cooperation with Syt. Syt1-SNARE and complexin-SNARE cooperate to activate synchronous release and regulate synchronous release after the AP has arrived at the synaptic terminal . Cav2  are the dominant channel subtypes for fast presynaptic transmitter. Cav2.2 (N-type) interacts with active zone proteins  and SNAREs to regulate the vesicle fusion. RIM-C2A and RIM-C2B bind the pore-forming subunit of N-type Cav channels in a Ca2+-independent manner that weakly interacts with the Cav1.2 , but do not interact with the Cav1.3 . Furthermore, RIM (C2 domain) enhances the interaction with syt-1 when intracellular Ca2+ concentration is increased. Removal of RIM domain heavily reduces the channel current and number of docking vesicles resulting in decreasing Ca2+ channel coupling with vesicle. The central PDZ-domain of RIM interacts with the C-terminal of presynaptic N- and P/Q-type Ca2+-channels, with no act on L-type Ca2+ channels. Deletion of RIM inhibits most neurotransmitter release due to impairing the synaptic priming and decreasing the Ca2+ channels localization in presynaptic membrane  induces gene variants of the C-terminal region (CTD) of Cav2.1. The two exons interact with RIM (1α and 2α), impair the binding of CTD with RIM and implied suppressive effect of RIM on voltage-dependent inactivation  channels are localized at the presynaptic terminal and trigger neurotransmitter release by enhancing presynaptic Ca2+ levels. Wu et al. (v2.3) Ca2+ channels contributed to about 26% of the total Ca2+ current during a medial nucleus of the trapezoid body presynaptic AP, but display a lower efficacy than other types of Ca2+ channels. R-type Ca2+ channels are also involved in fast synaptic excitation  and protein kinase C in rat cerebrocortical nerve terminals and not by intracellular Ca2+ release.Aplysia pleural sensory neurons are involved in the forms of presynaptic plasticity. The Aplysia Cau et al.  first deV3 family CaV3.1(α1G), CaV3.2(α1H), and CaV3.3(α1I) mediate T-type Ca2+ currents. T-type channels have been revealed to regulate neurotransmitter release in central, peripheral synapses and neuroendocrine cells that modulate basal neurosecretion close to resting potential with mild stimulations. Although T-type channels have no directly binding peptide (no synprint binding site), Cav3.2 channels interact with syntaxin 1A and SNAP-25. The interactions form nanodomains that can be regulated transiently and low voltages controlling neural activity and neuroendocrine. Interaction of T-type channels, secretory vesicles, and SNAREs form a nanodomains complex. T-type Ca2+ channels can directly interact with SNAREs (syntaxin 1A-Cav3.2-SNAP25) to control exocytosis. It is clear that T-type channels contribute to synaptic transmission in neurons and neuroendocrine cells under conditions of rest and mild stimulation. T-type Ca2+ channels are also involved in the development of a neuropathic pain. T-type Ca2+ channel subunit CaVα2δ interaction with the extracellular matrix protein thrombospondin-4 (TSP4) contributes to initiate, but not for the maintenance of excitatory synaptogenesis. Treatment with gabapentin blocks the early pain state but does not reverse the delayed state. It implies that early intervention with gabapentin may prevent the development of injury-induced chronic pain, one of the reasons is that CaVα2δ1/TSP4 initiates abnormal synapse formation  proteins. Gβγ negatively regulates the neurotransmitter release by inhibition of CaV2 (P/Q- and N-type) Ca2+ channels in synaptic terminals. Gβγ directly binds to CaV2.2α1 at the N-terminal45–55  produces a more potent effect , protein kinases  and Cae Figure .2+ channels and SNAREs  or PKC isozymes (βII or ε) are unable to potentiate Cav2.2 current in the presence of CaVβ subunits. Cavβ subunits complete suppression of the interactions between PKC and Ser/Thr sites of Cav2.2α1 subunits  are localized in presynaptic terminals that can phosphorylate both Cas Figure . PKC andThe phosphorylation of Core-conserved residues inside the SNARE domain can suppress vesicle fusion. Studies revealed that secretory protein VAMP8 phosphorylation by PKC at multiple residues in the SNARE domain mediated vesicle fusion, where protein kinase activation decreases and phosphatase activation increases the capacity of VAMP8  proteins are involved in neuronal Ca2+ signaling transmitter. The distance between voltage-gate Ca2+ channels and CaS for exocytosis determines the timing and probability of neurotransmitter release  and IQ-like motif (IM) of CaV2.1 contribute to facilitate and inactivate Cav2.1 channels. Mutation of the motifs of CBD and IM prevents synaptic facilitation. Nanou et al. , Visinin-like protein 2 (VILIP-2) and neuronal calcium sensor-1 (NCS-1) are the key CaS proteins for synaptic transmission. CaBP1 is highly expressed in the brain and retina, and co-localized in the CBD of Cav2.1α1  has been also shown to enhance synaptic facilitation. NCS-1 directly interacts with IQ-like motif and CBD site at the C-terminal domain of CaRH, JZ, YY and LZ contributed to the review of the literature, and editing of the manuscript. WW and ML wrote the draft manuscript. All authors read and approved the submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.