Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca Worldwide, approximately 20% of the irrigated agricultural land is affected by soil salinity . This le+ and Cl- in the cytosol of cells in the shoot, resulting in the inhibition of cellular processes and induces premature leaf senescence cyt) are involved in a variety of plant signalling pathways such as abiotic stress responses [2+]cyt follow a spatial and temporal pattern , inducing a stimulus-specific response cyt are sensed by Salt Overly Sensitive 3  and the binding of Ca2+ facilitates the interaction of SOS3 with SOS2  cyt cyt ,22. Earl[Na+]cyt , however2+ signalling pathway cyt present at any given time. It has been used previously to measure increases in [Ca2+]cyt to analyse the response of Arabidopsis seedlings to abiotic stresses such as drought, cold and salt stress cyt will be discussed.Here we show that the analysis of calcium signatures in response to salt stress might offer an opportunity to further investigate components of the salt signalling pathway. We provide evidence that calcium signatures evoked by salt treatment vary between the responsive ecotype Col-0 and the less-responsive ecotype C24. In addition, the importance of the temporal aspect of alterations in . The experiments were conducted in this depolarising base solution because of its simple composition as described previously cyt was performed exactly as has been described elsewhere [Seedlings were grown for 13 d to 15 d before developmentally similar seedlings were placed into luminometer cuvettes  containing 300 μL reconstitution solution [1.4 mM CaCleviously  and its eviously . TreatmeIn order to determine whether there was a significant difference in the response curves between Col-0 and C24 the following data analysis was performed.The response curves of three biological replicates for each ecotype and each treatment concentration was averaged.2+]cyt. The first peak was defined as the local maximum that occurred within the first 30 seconds after stimulus onset, while the second peak was defined as the local maximum that occurred between 30 and 120 seconds after stimulus application.The response curve was split into two temporal regions to separately identify two local peaks in cyt lasting approximately 20 s of similar magnitudes for both ecotypes was observed .The observed differences provide evidence that the early stages of the salt signalling pathway might differ in these ecotypes. The salt signalling pathway is triggered by a NaCl stimulus and initiates a signalling cascade, which results in the influx of Ca2+]cyt alterations in Col-0 and C24. To analyse whether it was possible to discriminate between the two ecotypes, the commonly-used linear discriminant analysis was performed. The base 10 logarithm of the amplitude of the second peak was plotted against the base 10 logarithm of the amplitude of the first peak, with the linear discriminant function drawn in the graph as a dotted line  was used to further analyse the peak behaviour of NaCl induced cyt is induced by the ionic component of NaCl, as it does not occur when the same strength of osmotic stress is applied using sorbitol. The results presented here support this hypothesis, by displaying the biphasic characteristics of NaCl induced [Ca2+]cyt alterations and monophasic sorbitol- and cold-induced [Ca2+]cyt alterations for the Col-0 ecotype  [2+]cyt signals along the root towards the shoot, which have recently been described using local NaCl stimuli to the root tip of Arabidopsis seedlings cyt signatures in Col-0 and C24. However, the molecular identity of the salt sensor, signal transmission molecules and the Ca2+ influx systems are poorly understood and it cannot be excluded that a completely different mechanism is underlying these differences in [Ca2+]cyt alterations. On the other hand, it is of great advantage that the genomes of Col-0 and C24 have been fully sequenced [Any of the three main stages in the early signalling pathway (salt sensing, signal transmission or Caequenced , making A high throughput genetic screen of a Col-0 x C24 population, based on the aequorin bioluminescence system, could lead to the identification of as yet unknown components in the early stages of the salt signalling pathway.


The [Ca2+] readout is not affected by dye concentration, light scattering, photobleaching, micro-viscosity, temperature, or the main known concomitants of cellular activity. In quiescent tissue, standard whole-cell configuration has little effect on resting [Ca2+] inside neuronal dendrites or inside astroglia dye-filled via gap junctions. Mapping basal [Ca2+] in neurons and astrocytes with submicron resolution unveils heterogeneous concentration landscapes that depend on age and preceding activity. The rich information content represented by such landscapes in acute slices and in vivo promises to unveil the hitherto unexplored, potentially fundamental aspects of brain cell physiology.Maintaining low intracellular calcium is essential to the functioning of brain cells, yet the phenomenology and mechanisms involved remain an enigma. We have advanced a two-photon excitation time-resolved imaging technique, which exploits high sensitivity of the OGB-1 fluorescence lifetime to nanomolar Ca •2+] and enables 100 Hz acquisition rateOGB-1 lifetime readout is specific to low [Ca•2+]Neurons and astrocytes maintain uneven landscapes of resting [Ca•2+] patterns in neurons and astroglia are age and activity dependentResting [Ca•2+]Astroglia in situ split into two sub-groups with respect to their resting [Ca Zheng et al. advance a fluorescence imaging technique enabling the monitoring of very low (nanomolar) calcium concentrations inside brain cells in situ with high resolution. The method uncovers rich information content of resting calcium landscapes inside neurons and astroglia. In neurons, release of neurotransmitters is controlled by presynaptic Ca2+ entry  is limited by the errors pertinent to the low signal-to-noise ratio, fluctuations in the dye concentration, focus drift, or photobleaching  exploits the fact that photon emission is a stochastic event occurring for up to several nanoseconds post-excitation, with a progressively declining probability. By registering a large number of emission events, FLIM thus restores the fluorescence signal decay time course (see below). This decay could be highly sensitive to molecular microenvironment; FLIM techniques have been emerging that use specific indicators to monitor nanometer-scale molecular interactions in live cells .2+ dyes, such as Oregon Green BAPTA-1 (OGB-1), is sensitive to free Ca2+ in the nanomolar range (2+] in a mouse model of Alzheimer’s disease (Because the fluorescence decay does not depend on how many dye molecules are imaged or how many photons are counted (these numbers will instead affect the measurement error), FLIM readout is inherently insensitive to the emission intensity or dye concentration. Consequently, it is also insensitive to focus fluctuations or light scattering, which all represent the key factors hampering fluorescence imaging in situ. It has also been found that the fluorescence lifetime of some commonly used Caar range . This fe disease . However disease , such as disease , tempera disease , or medi disease , validat2+-dependent lifetime of OGB-1 excited in two-photon mode is insensitive to physiological changes in pH, [Mg2+], [Zn2+], a ubiquitous neural protein (actin), temperature, or micro-viscosity. We implement a FLIM photon-counting procedure that boosts the readout stability, enabling a FLIM-mode image acquisition in situ at 100 Hz. We adapt this technique for high-sensitivity, high-resolution [Ca2+] monitoring in neurons and astroglia in acute brain slices and in vivo. Our case study unveils some previously unattainable, striking features of the intracellular Ca2+ homeostasis in the brain, suggesting a strong gnostic potential for the present technique.Here we show that the Cax2P = 800 nm) using standard solutions of buffered [Ca2+]  , compareximation B. Thus, moid fit C.2+] readout was affected by the common micro-environment factors in the intracellular milieu. First, the tests showed excellent stability for [Ca2+] evaluation over the wide range of pH  to ubiquitous cytoplasmic proteins in the muscle (2+] FLIM measure (2+ indicators. We thus deemed our OGB-1 FLIM readout (NTC) suitable for monitoring intracellular [Ca2+] in situ. Interestingly, [Ca2+] sensitivity of the OGB-1 fluorescence decay is not universal among Ca2+ dyes: the NTC readout of Fluo-4 was insensitive to [Ca2+] . Strikingly, in CA1 pyramidal neurons [Ca2+] dropped to 57 ± 8 nM almost immediately outside the pipette tip  but not correlated with the residual patch-pipette [Ca2+]  and also in conditions of OGB-1 single-cell bolus-patch loading .These observations indicate that principal neurons in situ possess a robust molecular machinery to maintain low [Ca2+] measured in readily identifiable somata of cortical neurons . In this protocol, the dye is taken up and subsequently hydrolyzed inside cells . Basal [rons see  can cope2+] map  in a rapid imaging sequence, with 10 ms temporal resolution , remained remarkably stable, both across the cell population . Consistent with previous observations  effect on the time-averaged [Ca2+] measure over a typical 50 s exposure  and was negatively correlated with the head volume  led to a significant reduction of [Ca2+] in dendritic shafts  and in spine heads  lasting for at least 5–10 min . This observation suggests the existence of a rapidly occurring use-dependent plasticity mechanism involving postsynaptic Ca2+ homeostasis.The observed developmental changes in dendritic [Ca5–10 min G and 4H.2+] changes during development. First, we found that resting astroglial [Ca2+] decreased dramatically within the week from P15 to P21 .While the average resting  nM E, albeit2+] sub-groups showed any spatial associations. Indeed, we found that basal [Ca2+] values were highly correlated between the nearest astrocyte neighbors  prompted us to ask whether these two  A. This i] levels B. It wou] levels .2+] and [Ca2+] dynamics in neurons and astroglia in situ at high resolution using two-photon excitation FLIM of the common Ca2+ indicator OGB-1. The FLIM-based [Ca2+] readout (NTC) provides unprecedented [Ca2+] sensitivity in the 10–200 nM range and is inherently insensitive to the dye concentration or depth of imaging in organized brain tissue. We have also found that this method of [Ca2+] measurement is insensitive to all major concomitants of intracellular microenvironment.In this study we have implemented and validated the method of monitoring low resting [Ca2+] essential for understanding cell physiology that relies on Ca2+ signals? The level of free Ca2+ results from a dynamic equilibrium between local Ca2+ entry, removal, and binding-unbinding with local Ca2+ buffers. Thus, it is a key reflection of the Ca2+ homeostasis machinery present in a given cell compartment. Correspondingly, changes in resting [Ca2+] could directly alter the availability (and thus buffering capacity) of Ca2+-free endogenous Ca2+-binding proteins; in physiologically relevant circumstances this relationship could be roughly proportional, even though changes in [Ca2+] and changes in the available buffer concentration are likely to occur on the nanomolar and micromolar scales, respectively  and a DOWN state  and 50–250 μm deep in vivo . We ensured that no contaminating fluorescence signal was collected from damaged tissue near the slice surface, and no autofluorescence was detected at these depths (before applying OGB-1). Importantly, a short-pass 700 nm filter was placed in front of the detector to block out any escaped light from the laser source. The imaging system was based on a Biorad Radiance 2000 coupled with SPC-830 TCSPC Becker & Hickl imaging module under an Olympus LumplanFI 60× water immersion objective (NA 0.9) or an Olympus FV1000 coupled with Picoquant Picoharp 300 TCSPC system under Olympus XLPlan N 25× water immersion multi-photon objective (NA 1.05). Various digital zooms (2×–15×) were used to allow imaging of different cellular structures. The two-photon laser source was a Newport-Spectraphysics Ti:Sapphire MaiTai laser pulsing at 80 Mhz, with a pulse width of ∼220 fs and a wavelength of 800 nm optimized for OGB-1 excitation. The laser power was kept below 8 mW under the objective at all times for slice preparations to minimize phototoxic damage. The laser power was also minimized for in vivo imaging according to depth, where a maximum ∼70 mW power under the objective was used for imaging depth at ∼250 μm depth.t,x,y,T) tensor representing x-y images with a distribution of nanosecond delay time (t) of photons at each pixel over the frame duration (T), which could also include z stack information. Average image acquisition times were 30–300 s (maximum laser exposure time of <100 s) depending on the total photon count, and the maximum photon count rate was on average <105 s−1, which is well below the effect of photon pile-up . To monitor possible [Ca2+] fluctuations, we minimized the sampled image area to approximately 6 × 3 μm  and a digital size of 100 × 50 pixels  so that a 10 Hz image acquisition rate could be achieved. Acquisition time of 10 s for each session was used, with >30 s resting period between each session, to minimize phototoxic damage. Because of relatively low photon counts per pixel, the photon data were accumulated over the entire image from  tensor into  matrix before the data analysis was used to estimate Ca2+ concentrations.Fluorescence images were acquired at a laser line scanning rate of 500 lines per second and stored as a 256 × 256 × 256 × n  photon counting data at any given pixel or ROI. Physical hardware (Becker & Hickl HPC-CON unit) can also be used to split the same signal stream from the photo-detector into digital  and analog (photon intensity) signal streams so that intensity and FLIM data can be acquired simultaneously in the same imaging system.2+ calibration buffer kit manual. However, to match the Ca2+ buffering dynamics to that of OGB-1 more closely, the standard 10 mM chelating agent EGTA was replaced with 10 mM BAPTA, and the solution constituents were replaced with our experimental intracellular solution (see below). pH was adjusted using KOH, and the KCl concentration in the intracellular solution was adjusted accordingly to keep ion constituents in the solution unchanged. The estimated [Ca2+] was therefore slightly different from the standard Invitrogen’s calibration set and was finely adjusted using Chris Patton’s WEBMAXC program at Stanford University (http://web.stanford.edu/∼cpatton/webmaxcS.htm). In the acidity control tests, pH was altered using KOH. [Mg2+] and [Zn2+] were changed using MgCl2 and ZnCl2, respectively, whereas other constituents were adjusted to maintain the original composition of the intracellular solution. Actin protein calibration was adjusted using Actin from Bovine Muscle (Sigma A3653), temperature was varied (from 19°C to 37°C) using a Scientific Systems Design PTC03 in-line heater, and solution viscosity was varied by adding 70 kDa Dextran to ACSF or internal solution.The OGB-1 calcium calibration protocol was similar to the standard calibration method provided by the Invitrogen Ca2+] using an appropriate calibration function obtained through direct OGB-1 calibration for intracellular solutions of clamped [Ca2+], as further explained in the text.The fluorescent decay time course was first normalized to its peak value and then integrated (area under curve) over 9 ns post-peak A. The re2+]-mapping pixel values were spatially averaged using 5–10 nearest-neighbor pixels to ensure that the FLIM decay traces had at least 10 counts near the tail. Any pixel that did not satisfy such criteria was not calculated as a valid data point and discarded (depicted in images as blank pixels). All the statistics and metrics for individual cellular compartments were, however, obtained from the raw data matrices, not from the smoothed mapped images. To maximize the measurement accuracy and reliability for individual selected cell compartments, we used photon count information within this region only and averaged the data to give an overall [Ca2+] estimate for that compartment.For illustration purposes,  data point was sufficient to meet the analysis criteria for reliable [Ca2+] readout (see below). In our conditions we thus were able to increase the FLIM acquisition frequency to 100 Hz.To enable monitoring of rapid Ca3 26, KCl 2.5, NaH2PO4 1.25, MgCl2 7, CaCl2 0.5, glucose 11, ascorbic acid 1.3, and sodium pyruvate 3 (osmolarity 300–310 mOsM), stored in the slicing solution at 34°C for 15 min, and transferred for storage in an extracellular solution containing (in mM) NaCl 125, NaHCO3 26, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3, CaCl2 2, and glucose 16 (osmolarity 300–305 mOsm). All solutions were continuously bubbled with 95% O2/5% CO2. Slices were allowed to rest for at least 60 min before recordings started.Acute hippocampal transverse slices (350 μm thick) were prepared from P7–P8, P15–P16, and P21–P24 Sprague-Dawley rats, in full compliance with the national guidelines, the European Communities Council Directive of 24 November 1986, and the European Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes. Slices were prepared in an ice-cold slicing solution containing (in mM) NaCl 60, sucrose 105, NaHCOstratum radiatum astrocytes were performed in a submersion-type recording chamber. Slices were superfused with an extracellular solution containing (in mM) NaCl 125, NaHCO3 26, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3, CaCl2 2, and glucose 16 (osmolarity 300–305 mOsm), continuously bubbled with 95% O2/5% CO2. Whole-cell recordings were obtained with patch pipettes (3–5 MΩ) with an intracellular solution containing (in mM) KCH3O3S 135, HEPES 10, Tris-phosphocreatine 10, MgCl2 4, Na2ATP 4, and Na3GTP 0.4 . The cell impermeable Ca2+ indicator OGB-1  was added to the internal solution. CA1 pyramidal cells were held at −70 mV. Protoplasmic astrocytes located in the stratum radiatum were identified by their small soma size, low resting potential (<−80 mV), and low input resistance (<10 MΩ). Astrocytes were held in voltage clamp at their resting potential or in current clamp.Whole-cell patch-clamp recordings of CA1 pyramidal cells and 2+] FLIM measurements were carried out across the dendritic tree several minutes after the re-seal to minimize diffusion escape of the dye.In single-cell bolus-loading experiments, cells were held in whole-cell mode for only 1–3 min, after which the pipette was carefully withdrawn until an outside-out configuration was obtained to allow the somatic membrane to seal safely. [CaAll in vivo animal experiments were conducted using male Sprague-Dawley rats (p27–P33) in accordance with the European Commission Directive (86/609/EEC) and the United Kingdom Home Office (Scientific Procedures) Act (1986).2-enriched room air using a positive pressure ventilator with a tidal volume of ∼1 ml/100 g and a ventilator frequency similar to the resting respiratory rate (∼60 strokes/min). The animal was then placed in a stereotaxic frame. The skin overlying the skull was removed, and a small craniotomy (∼2 mm2) was made in the parietal bone above the somatosensory cortex. Cortical astrocytes were labeled with sulforhodamine 101 (SR101) and OGB-1. The solution containing OGB-1 (1 mM) and SR101  in artificial cerebrospinal fluid  was delivered (volume 1.5 μl) via a glass micropipette to the targeted area of the right primary somatosensory cortex, immediately caudal of the coronal suture . The exposed surface of the cortex was then covered with 1% agarose and protected with a glass coverslip secured to the skull using acrylic dental cement.Young male rats (100–120 g) were anesthetized with urethane  following isoflurane (5% in air) induction. Adequate anesthesia was ensured by maintaining stable levels of the arterial blood pressure and heart rate showing lack of responses to a paw pinch. The femoral artery and vein were cannulated for measurement of the arterial blood pressure and administration of anesthetic, respectively. The trachea was cannulated, and the animal was ventilated with OPO2, PCO2, and pH of the arterial blood were periodically measured using a RAPIDLab 348EX blood gas analyzer , and the ventilation parameters were adjusted to keep these variables within the physiological ranges. Two-photon excitation for FLIM acquisition was carried out as described below using a Newport-Spectraphysics Ti:Sapphire MaiTai laser, Olympus FV1000 with XLPlan N 25× water immersion multi-photon objective (NA 1.05), and Picoquant Picoharp 300 TCSPC. Acquisitions were carried out at a depth between 50 and 300 μm from the cortical surface.Throughout surgical preparation and imaging, body temperature was maintained at 37.0°C. Vs) estimate  is that the fluorophore concentration and excitation laser power are uniform within the distal dendritic branches and nearby spines monitored in the same focal plane. The cytosolic volume of the compartment is therefore proportional to the fluorescent intensity observed, i.e., Vs / Vd = Is / Id, where index d stands for dendritic shaft values. The ROI (in the x-y plane) for Vd in the dendritic shaft was, however, selected to be as small as the smallest spine in the same image. This corresponded to the dendritic shaft volume, which was smaller than the shaft diameter, thus providing 100% sampled emission represented by the fluorophore, also mitigating the effect of boundaries and volume fractions (Vd represented the average fluorescence intensity over the depth of the system’s point spread function (PSF), whereas Vs represented a fraction of this intensity over the same depth, thus directly reflecting the structure volume. Because the system PSF was unchanged throughout the experiments, Vs / Vd provided a fully consistent and unbiased relative volume measure. To arrive at the absolute volume scale, we used the characteristic PSF depth at 800 nm .K.Z. designed and implemented the FLIM-based method, including imaging and analysis protocols; L.B. carried out patch-clamp and imaging experiments in neurons and glia; T.P.J. carried out some patch-clamp and imaging experiments in neurons; A.V.G, J.R., K.Z., and C.K. designed and carried out in vivo experiments; and D.A.R. narrated the study, which was subsequently contributed to by all the authors.


To investigate the molecular mechanism of how these two ligands induce different behaviors of the receptor, we built two systems containing the 5′-GNTI-κOR complex and the 6′-GNTI-κOR complex, respectively, and performed molecular dynamics simulations of the two systems. We observe that transmembrane (TM) helix 6 of the κOR rotates about 4.6o on average in the κOR-6′-GNTI complex. Detailed analyses of the simulation results indicate that E2976.58 and I2946.55 play crucial roles in the rotation of TM6. In the simulation of the κOR-5′-GNTI system, it is revealed that 5′-GNTI can stabilize TM6 in the inactive state form. In addition, the kink of TM7 is stabilized by a hydrogen bond between S3247.47 and the residue V691.42 on TM1.The  Among the three subtypes, the κOR is a promising drug target for alleviating pain with a possible lower abuse potential45κOR subtype represents an excellent target to treat neurological disorders789101112κOR have been developed to treat related disordersκOR have proved to be effective on curing depressant and anxiolytic diseases151617Opioid receptors (ORs) are important members of G-protein-coupled receptors (GPCRs) and the main targets for analgesics12κOR, 5′-GNTI  and 6′-GNTI  are an interesting pair  simulations of systems with the κOR in the apo state and with 5′-GNTI-κOR and 6′-GNTI-κOR complexes, respectively, to provide active and inactive models of the GPCR based on the action of the so-called “molecular switches“32κOR was observed to rotate about 4.6o on average in the κOR-6′-GNTI complex. Helices TM7 and TM2 highly responded to the activation signal in 6′-GNTI systems too. In the simulation of the κOR-5′-GNTI system, 5′-GNTI could stabilize TM6 in the inactive state form.To explicate the activation mechanism of the 3.32 in each complex. The phenolic hydroxyl group of both ligands interacts with H2916.52 bridged by two water molecules and the cyclopropyl group interacts with W2876.48. Meanwhile, Y1393.33 and Y3207.43 also contribute to the stabilization of the two ligands at the orthosteric site. Because of the difference in the substitution site of the guanidinium group in 6′-GNTI and 5′-GNTI, different interaction patens were observed when the two ligands bind to the κOR. The guanidinium group of 5′-GNTI is located in the cavity formed by the residues on TM6 and TM7 and forms three hydrogen bonds with E2976.58, whereas the guanidinium group of 6′-GNTI is positioned in a cavity formed by the residues on TM5 and TM6 and forms only one hydrogen bond with the residue E2976.58. In addition, the docking poses of the two GNTIs are almost identical with the conformation of NTI adopted in the crystal structure of the NTI-δOR complex. With the structures of 5′-GNTI or 6′-GNTI in the orthosteric pocket of the κOR and the κOR in the apo state, we built three systems, 5′-GNTI-κOR, 6′-GNTI-κOR, and the κOR in the apo state, to be used for the following simulations.The docking poses of 6′-GNTI and 5′-GNTI are almost identical with each other as shown in κOR with respect to its crystal structure range from 2.0 to 3.6 Å  values in the simulations . The RMSto 3.6 Å . The ligto 3.6 Å . In addito 3.6 Å . IL κOR. The binding of GNTIs causes large conformational change of TM3 in the GNTIs−κOR complexes as shown in our simulations. In the simulation of the 5′-GNTI-κOR complex, we observed a clockwise rotation of TM7 if viewed from the extracellular side  and in the apo κOR (6.1%). A recent study on CRF1R also proved the significance of the kink in explaining the antagonistic mechanismA detailed analysis of the simulation results reveals that the kink is stabilized by a hydrogen bond formed between S324 V691.42 . The occβ1-adrenergic receptor and δOR2842δOR, the sodium ion is positioned around the negatively changed D2.50, and coordinated by two polar residues (N1313.35 and S1353.39) and two water moleculesμOR suggested that a sodium ion penetrates into the allosteric pocket from the extracellular side of the receptor to perform its allosteric effects on the receptorκOR have also been described in the latest work of Filizola et al.The role of the sodium ion in the function of some GPCRs has become an attractive topic recently. The crystallographic structure of the adenosine A2A receptor (A2AR) at an ultra-high resolution provides us with the first evidence of the binding of the sodium ion to the GPCR, followed by the crystallographic structures of the κOR, the sodium ion is not co-crystallized with the κOR. We thus placed a sodium ion in the allosteric pocket of the κOR by referring to the position of the co-crystallized sodium ion in the crystal structure of the NTI-δOR complexIn the crystal structure of the 2.50 increased significantly during 180–300 ns in the simulation of the 6′-GNTI-κOR system due to the conformational change of D1052.50 , was obtained from an engineered κOR mutant protein where part of the intracellular loop between transmembrane helices TM5 and TM6, was replaced by T4 lysozyme (T4L)κOR dimer, we removed the T4L from the mutant receptor structure, the monomer B and other unnecessary parts. Then we reconstructed the loop of the remaining chain A by adding the missing residues S262 and T302–S303–H304–S305–T306 using the loop refinement protocol in Discovery Studio 3.5; 10 loops were generated and the most reasonable one was chosen for receptor construction. According to the latest crystal structure of inactive NTI-complexed δOR, ion Na+ was exactly located at the allosteric pocket positionThe published crystal structure of human inactive JDTic-bound κOR structure was then imported into the Schrödinger software package. The protein structure was prepared with Protein Preparation Tool (ProPrep) in the Schrodinger 2012 suit software. Asn, Gln, and His residues checked for protonated states automatically in ProPrep. Hydrogen atoms were added into the κOR crystal structure at the physiology pH environment by the PROPKA tool in Maestro with optimized hydrogen-bond network. No non-standard protonation state of the amino acids was found.The above three-dimensional integrated δOR, κOR-selective antagonist 5′-GNTI and κOR-selective agonist 6′-GNTI were sketched in Maestro and subjected to a Monte Carlo Μultiple Minimum conformational search using the OPLS_2005 force field and water as implicit solvent  model). The output conformations were used as the starting point for the docking experiments.By referring to the conformation of ligand NTI in crystal structures of κOR was generated by 20 Å around residue D1383.32 with the Receptor Grid Generation module, and docked with Glide Docking module (Glide 5.8)49The pocket Grid file of the κOR complexes, one 6′-GNTI-κOR complex and one apo-κOR system, were built for the simulations. A POPC  bilayer with the surface area of 75 × 75 Å2 on the X-Y plane was generated under Charmm36 force field by VMD program (Version 1.9.1). For each system, the receptor was first embedded into the POPC bilayer using our in-house program pre-aligned in the OPM (Orientations of Proteins in Membranes) database5152533 were used solvate the protein. Lipid molecules within 0.85 Å of the heavy atoms on the protein structure and water molecules in the bilayer were removed. 51 Na+ and 59 Cl- ions were used to produce neutral systems with 0.15 M NaCl in the water phase of GNTI systems, whereas the number of Cl- ions was 57 in apo-κOR system. We described the protein using CHARMM36 force field with cmap correction.Two 5′-GNTI-Ligand force field was generated by Paramchem webserver, a program coupled with CHARMM Force field5556κOR system was performed for 100 ns, all the other systems were performed for about 600 ns using the NPT ensemble with the Parrinello-Rahman pressure coupling and the Nose-Hoover temperature coupling methods.All simulations were performed using Gromacs V.4.6.5RMSD and RMSF calculations, ligand cluster statistics, hydrogen bond analysis, angle and distance evolutions were produced by the program Gromacs. The interval time of trajectory calculations, including RMSD, RMSF, angle, distance and principal component analysis, was 500 ps in 600 ns systems, whereas that in 100 ns 5′-GNTI systems was 100 ps. RMSD values were calculated through comparing to the initial simulation conformations. Principal components analysis was carried on through the g_covar tool. All the smooth curves in κOR, structural clustering was performed using the g_cluster tool in Gromacs with the linkage algorithm based on the RMSD of each ligand molecule after alignment of the Cα atoms in the transmembrane helices in each frame to the starting structureTo determine the sub-state conformations that close ligands bound to α of V2856.46, P2896.50 and F2936.54; The angle between I2946.55 and TM6 was measured with atom Cδ and Cα of I2946.55 and atom Cα of W2876.48, while that angle between E2976.58 and TM6 was computed with atom Cδ and Cα of E2976.58 and atom Cα of F2936.54; The kink angle of TM7 was measured with atom Cα of Y3207.43, P3277.50 and A3317.54; The angle between D1052.50 and TM2 was measured with atom Cγ and Cα of D1052.50 and atom Cα of I982.43.Angle analysis was carried on by g_angle tool in Gromacs. The specific input parameters were as follows. The kink angle of TM6 was measured with atom Chttp://www.igs.cnrs-mrs.fr/elnemo/index.html), a web interface to the elastic network model-based NMA.NMA was conducted using the ElNemoHow to cite this article: Cheng, J. et al. Molecular switches of the κ opioid receptor triggered by 6′-GNTI and 5′-GNTI. Sci. Rep.6, 18913; doi: 10.1038/srep18913 (2016).


In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling.YEMK. In duct cells, CDCA caused reversible increase in the intracellular Ca2+ concentration [Ca2 +]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca2+ signals, but could stimulate Na+/Ca2+ exchange in some conditions.Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.032+]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca2+ conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [CaThe online version of this article (doi:10.1186/s12964-015-0107-9) contains supplementary material, which is available to authorized users. In recent years there has been a growing interest for the role of bile acids as signalling molecules in many cells/organs. Several types of bile acids receptors have been identified, but some modulatory functions of bile acids remain unexplained. Here, we show that bile acids, in particular chenodeoxycholic acid, causes significant release of ATP from exocrine pancreatic cells. Extracellular ATP can then via purinergic receptors regulate or co-regulate epithelial functions, such as pancreatic duct secretion, which is important for normal digestive processes. Our study brings novel insights into regulation of pancreas functions. Moreover, we propose that purinergic signalling should be taken into consideration in other cells/organ types, as it could potentially explain some of the multifaceted effects of bile acids.3− and fluid secretion . T. T4]. TG. T. T4].ecretion –14.2+-independent cation currents i, in pancreatic acini. This is due to inhibition of the sacro/endoplasmatic reticulum Ca2+-ATPases (SERCA), release of Ca2+ from ER and acidic stores and granules, increased Ca2+ influx, and acini show cellular acidosis, enzyme activation and mitochondrial malfunction, which can eventually lead to development of (biliary) acute pancreatitis i. imaging. Our study shows that CDCA indeed caused a very fast release of ATP from exocrine pancreatic cells via both non-exocytotic and vesicular pathways. Furthermore, a significant part of the CDCA effect on Ca2+ signalling is mediated via purinergic signalling. We also demonstrate the presence of the TGR5 and FXR receptors in human pancreatic ducts and show that TGR5 receptor can prevent intracellular Ca2+ overloads possibly by stimulation of the Na+/Ca2+ exchanger.Considering that BAs and purinergic signalling appear to have similar paracrine effects on ion and fluid transport in exocrine pancreas, we hypothesized whether there was any interaction between these two intra-ductal regulatory systems. Therefore, we designed a study to test whether BAs, i.e., chenodeoxycholic acid (CDCA) and its glycine and taurine conjugated forms (GCDCA and TCDCA) can affect ATP release, and whether CDCA also influence intracellular ATP levels. A further aim was to elucidate whether BA signalling involves TGR5 or FXR receptors in the ATP release process and/or P2 receptors and downstream Cae, by 4.2 ± 0.8 nM (n = 4) above the basal level. TCDCA (1 mM) had no effect on ATP release. In contrast to GCDCA and TCDCA, CDCA (0.3 mM) caused a fast and substantial ATP release in Capan-1 cells  for Capan-1 and 614 ± 79 nM (n = 5) for AR42J cells. These data show that CDCA can increase extracellular ATP concentrations by a factor of 100–1000 above the baseline. In all the following experiments, Capan-1 cells were stimulated with 0.3 mM CDCA and AR42J cells with 0.5 mM CDCA.ATP release on the whole organ level is difficult to detect due to action of membrane-bound and soluble nucleotidases , 38. Thells Fig. . Therefolls Fig. . Furtherl. TCDCA  mM had nl. TCDCA  mM had nSince Capan-1 cells are a model of human pancreatic duct epithelium, it was relevant to investigate whether ATP release is polarized, i.e., whether it occurs preferentially across the luminal or the basolateral membrane. Capan-1 cells were cultured as polarized monolayers, and CDCA was administered luminally or basolaterally. After the luminal stimulation with 0.3 mM CDCA Fig. , there w+-ATPase, ATP release was significantly reduced to 61 ± 12 nM (n = 5). Inhibition of CDCA-evoked vesicular ATP release was also observed in the presence of N-Ethylmaleimide, an inhibitor of vesicle fusion, which markedly supressed ATP release to 9 ± 3 nM (n = 4). In addition, reducing vesicular transport from ER to Golgi by brefeldin A also decreased the ATP release to 55 ± 11 nM (n = 8). In another series of experiments, we tested the effect of non-vesicular transport inhibitors  compared to control of 76 ± 15 nM (n = 10). When cells were treated with probenecid, there was a significant decrease in ATP release to 24 ± 9 nM (n = 7). The pannexin-1 inhibitor, 10Panx, tended to decrease ATP release to 47 ± 15 nM (n = 7), but no statistical significance was reached. Two P2X7 receptor antagonists significantly inhibited the ATP release to 19 ± 5 nM  and 25 ± 5 nM . Taken together, present data indicate that both vesicular and non-vesicular mechanisms are involved in CDCA-induced ATP release from pancreatic epithelial cells.In order to identify the pathways involved in CDCA-induced ATP release, Capan-1 cells were incubated with vesicular and non-vesicular transport inhibitors. Fig. ors Fig. . Data pri. We used the Magnesium Green (MgGreen), an indirect ATP sensor, as it increases in fluorescence when ATP concentrations decrease and the released Mg2+ can bind to the fluorophore. After CDCA stimulation, there was a fast and transient increase in the MgGreen F/F0 fluorescence ratio to 1.53 ± 0.21 in AR42J (n = 3), and then the ratio decreased but remained elevated by about 0.2 units above the basal value , correlating with the peak and plateau for MgGreen . Fig. i was not significantly changed and remained at 2.47 ± 0.32 mM calculated per cell; (n = 8) and 2.22 ± 0.5 mM (n = 7) compared to their respect controls of 2.72 ± 0.38 mM and 2.46 ± 0.45 mM (n = 7). Furthermore, the long-term exposure of Capan-1 to 0.3 mM CDCA did not cause significant changes in ATPi concentrations, i.e., 2.08 ± 0.24 mM compared to control 1.85 ± 0.18 mM (n = 10). For AR42J cells, stimulation with 0.5 mM CDCA . However, after 12 min there was a tendency, though not significant, of lower intracellular ATP levels  with CDCA compared to their respective controls . Furthermore, after 24 h incubation of AR42J with 0.5 mM CDCA, there was a significant decrease of ATPi to 0.66 ± 0.06 mM compared to the control 1.3 ± 0.08 mM (n = 10).The above ATPied Fig. . ATPi coeen Fig. . AdditioDCA Fig.  caused A2+]i in pancreatic cells, but it is not clear which receptors are involved (see Introduction). Since we observed that CDCA induced ATP release, we hypothesized that the intracellular Ca2+ responses could be due to effect of released ATP on P2 receptors, which are well established regulators of stimulated Ca2+ influx and/or Ca2+ release in pancreatic ducts. Therefore, [Ca2+]i was monitored in Capan-1 cells in a chamber perfused with physiological solution containing CDCA followed by ATP . Similar slow and small responses to other BAs were also reported for pancreatic acinar cells and cholangiocytes [2+]i increase . Further experiments were conducted without perfusion, in order to minimize mechanical stimulation and amount of inhibitors used. In standing bath CDCA evoked similar Δ[Ca2+]i response as in the perfused conditions, but the signal was delayed , perhaps because P2 receptors were already desensitized by CDCA-induced ATP release as ATP was not washed away during perfusion. Therefore, for the following studies with P2R inhibitors, we conducted separate experiments for the ATP and CDCA stimuli.Bile acids are reported to increase i response from 937 ± 88 nM (n = 11) to 232 ± 47 nM (n = 9). The antagonists also caused significant inhibition of CDCA stimulated Δ[Ca2+]i . These data indicate that the Δ[Ca2+]i response evoked by the bile acid could be a result of P2 receptor stimulation by CDCA-induced ATP release. Thapsigargin, the inhibitor of SERCA [2+ to intracellular stores. Thapsigargin induced a small Δ[Ca2+]i increase after CDCA by 70 ± 17 nM (n = 5) but a large increase after ATP, i.e., 971 ± 143 nM, (n = 6). P2R inhibitors did not have effect on intracellular Ca2+ response induced by thapsigargin after ATP .Figure of SERCA , was addATP Fig. . InteresATP Fig. , Δ[Ca2+]It is not known whether TGR5 and FXR receptors are expressed in the human pancreatic ducts and could account for the effects seen in our study. Also from studies on animal pancreatic tissue, it is not certain whether TGR5 is expressed in ducts , 29. We In another series of experiments we investigated whether Capan-1 cells express the nuclear type of bile acid receptor, FXR, and whether its stimulation might induce ATP release. We indeed found the transcript for FXR in Capan-1 cells using RT-PCR Fig. . However2+ efflux pathways. Our hypothesis was that the TGR5 receptor could be involved in these effects and we tested this in the following experiments. First, intracellular Ca2+ stores were emptied by thapsigargin in low Ca2+ medium .Above we observed that CDCA appeared to protect Capan-1 cells from thapsigargin effects Fig. . This coium Fig. . Thereaf nM Fig. , which l2+]i caused by CDCA could involve Ca2+ efflux via the Na+/Ca2+ exchanger (NCX), which is expressed in duct epithelia i, and the fluorophore can also bind Ca2+, the MgGreen signals can have several components. In addition, in AR42J cells there was a slow increase in MgGreen fluorescence occurring long after Ca2+ peak. There was a similar slow effect of CDCA on AT1.03YEMK ratio, as well as a change in the lifetime constants of the sensor. These data together indicate that there was a decrease in ATPi. Using luciferase assay, we find that in the first minute after CDCA stimulation acinar cells release significant amount of cellular ATP  caused ATP depletion in pancreatic acinar cells and colon epithelial cells, presumed to be due to inhibited metabolism , 64. SinATP Fig. , though say Fig. . This desay Fig. , which antration .2+]i in many cells, including pancreatic cells and mechanisms include increased release from ER, inhibition of SERCA and increased Ca2+ entry [s protein and leads to stimulation of adenylate cyclase and cAMP signalling [2+ [2+ transients in many cells and BA-stimulated amylase release is relatively undisturbed [2+ signalling. Since GPBAR-A had no effect on [Ca2+]i in our duct cells expressing the receptor, it seems that TGR5 receptor is not involved in initiating simple Ca2+ transients in given experimental conditions. We propose that CDCA-evoked ATP release leads to P2 receptor activation and thereby increases in [Ca2+]i. Indeed, CDCA-stimulated Ca2+ increase was inhibited by a cocktail of P2R antagonists . Our study did not address the question whether BAs via activation of FXR and TGR5 could regulate purinergic receptor expression on a longer time-scale.It is extensively documented that BAs (0.1 – 1 mM) cause an increase in i stress conditions in pancreas. It has been shown that CDCA can protect cells when ER is depleted of Ca2+ [2+]i and that the GPBAR-A was similarly effective , it can transport Ca2+ in or out of the cell, depending in electrochemical potential, and this could vary depending on the cell type and its stimulation.The differences in the thapsigargin induced Ca of Ca2+ . We obseive Fig. . Others -one  dexamethasone (50 nM). Cells were pre-treated/incubated with inhibitors and fluorescent indicators as follow: bafilomycin A1 (1 μM), N-Ethylmaleimide , brefeldin A , pannexin inhibitor 10Panx , gadolinium chloride , probenecid (500 μM), P2X7 inhibitors: AZ10606120  and A438079 , pyridoxal phosphate-6-azo tetrasodium salt hydrate , suramin 250 μM, Magnesium Green indicator , Fura-2 AM calcium indicator , pluronic F127 (Molecular Probes- Life Technology), ATP kit SL 144–041 (BioThema), cell counting kit-8 .All chemicals were purchased from Sigma-Aldrich unless otherwise specified. In present study following chemicals were used: chenodeoxycholic acid , glycochenodeoxycholic acid , taurochenodeoxycholic acids , ATP (100 μM), digitonin (50 μM), thapsigargin (1 μM), 4-methyl]-6-(2-fluorophenyl)-4,5-dihydro-pyridoi changes, Capan-1 cells were perfused with 5 mM Na+ buffer, where the rest of NaCl was substituted with 140 N-Methyl-D-Glucamine (NMDG) titrated with HCl. Fura-2 recording was performed on a system described earlier [in situ to calcium concentrations based on formula described by Grynkiewicz [d for Fura-2: 224 nM.Capan-1 cells were incubated with Fura-2 AM in the presence of 0.02 % pluronic F127 and probenecid (0.25 mM) in physiological buffer for 30 min. For perfusion experiments the perfusion rate was 1 ml/min. For standing bath (non-perfused) conditions cells were incubated for 25 min with or without P2R antagonists. In some experiments, Capan-1 cells were stimulated with Thapsigargin in nominal Ca earlier . Fura-2 nkiewicz  with Kd RNA was isolated using RNeasy Mini Kit (Qiangen 74104) following the manufacturer’s instructions. RT-PCR was analysed with QIAGEN OneStep RT-PCR Kit (210212) with amplification parameters as follows: one cycle at 50 °C for 30 min and one cycle at 95 °C for 15 min followed by 37 cycles at 95 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s, and one final cycle at 72 °C for 10 min. The following primers were designed using Primer BLAST and used for TGR5 amplification: human TGR5 forward 3′ TCCTGCCTCCTCGTCTACTT 5′ human TGR5 reverse 3′ GGTAGGGGGCTGGGAAGATA 5′(247 bp), human FXR forward 3′AGAGATGGGAATGTTGGCTGAA 5′ human FXR reverse 3′ GTGAGTTCAGTTTTCTCCCTG 5′(186 bp), rat TGR5 forward 3′ GCTACTGGAGTGGTAGGCAG 5′ rat TGR5 reverse 3′ TCAGTCTTGGCCTATGAGCG 5′(225 bp). All primers were synthesised by TAG Copenhagen A/S (Denmark).Protein lysates were prepared by adding lysis buffer  containing protease inhibitor. Cell lysates were centrifuged at 15,000 g for 15 min at 4 °C. To obtain the membrane microdomain enriched samples the lysate was centrifuged at 200,000 g for 1 h (Beckman Ultracentrifuge Ti 70.1 Rotor) . WesternAR42J cells were grown on glass coverslips  and Capan-1 cells were seeded on collagen coated Snapwells. The cells were gently washed with physiological PBS and fixed in 4 % paraformaldehyde in PBS for 15 min, treated with 0.1 M TRIS-glycine (pH 7.4) for 15 min, and then rinsed in PBS and permeabilized for 10 min in PBS with 0.5 % TritonX-100. Cells were blocked with 10 % BSA in PBS for 45 min and then incubated with TGR5  for 1.5 h. Slides were washed for 10 min and then incubated 1 h with 1:400 goat anti-rabbit secondary antibody conjugated to Alexa 488 (Life Technology). For nuclear staining, DAPI was used (1:400) and mounted with DAKO fluorescent mounting medium. Slides were viewed using a 40X N.A 1.3 objective with TCS SP 5X.t test was applied. For multiple conditions, one-way ANOVA with Bonferroni’s Multiple Comparison Test was used. P < 0.05 was considered statistically significant. For FLIM-FRET analysis and statistics see above.Data are shown as the mean values ± S.E.M. To test the statistical significance between two conditions, unpaired two-tail Student’s


In this study, we investigated whether activation of the canonical transient receptor potential (TRPC) 6 channels and successive [Ca2+]i elevation alter neonatal GMC survival. Hyperforin (HF)-induced TRPC6 channel activation increased [Ca2+]i concentration, inhibited proliferation, and triggered apoptotic cell death in primary neonatal pig GMCs. HF-induced neonatal GMC apoptosis was not associated with oxidative stress. However, HF-induced TRPC6 channel activation stimulated nuclear translocation of the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). HF also increased cell death surface receptor Fas ligand (FasL) level and caspase-8 activity in the cells; effects mitigated by [Ca2+]i chelator BAPTA, calcineurin/NFAT inhibitor VIVIT, and TRPC6 channel knockdown. Accordingly, HF-induced neonatal GMC apoptosis was attenuated by BAPTA, VIVIT, Fas blocking antibody, and a caspase-3/7 inhibitor. These findings suggest that TRPC6 channel-dependent [Ca2+]i elevation and the ensuing induction of the calcineurin/NFAT, FasL/Fas, and caspase signaling cascades promote neonatal pig GMC apoptosis.Glomerular mesangial cell (GMC) proliferation and death are involved in the pathogenesis of glomerular disorders. The mechanisms that control GMC survival are poorly understood, but may include signal transduction pathways that are modulated by changes in intracellular Ca First, we examined whether HF stimulates Ca2+ release from intracellular Ca2+ stores in the cells. In the absence of extracellular Ca2+, HF did not alter basal [Ca2+]i in the cells  elevated [Ca2+]i by 64.4 ± 9.2 nM  generation in control and neonatal pig GMCs treated with HF. Confocal microscopy showed that pyocyanin, a superoxide inducer stimulates superoxide generation in neonatal pig GMCs . By conthe cells . Next, whe cells . By conthe cells . These f2+]i elevation via TRPC6 channels stimulates nuclear translocation of NFATc1 in neonatal GMCs, we studied the localization of a GFP-NFATc1 cDNA clone that was transiently transfected into neonatal pig GMCs. Unlike DMSO (control)-treated cells, GFP-NFATc1 showed predominant nuclear localization in HF-treated cells , BAPTA, and inhibitors of NFAT, Fas, and caspase-3/7. As shown in 2+]i elicited by HF-induced TRPC6 channel activation, and sequential stimulation of the calcineurin/NFAT, FasL/Fas, and caspase signaling pathways promote neonatal GMC apoptosis -dependent serine/threonine phosphatase18192+]i, the Ca2+-CaM complex activates calcineurin. NFAT proteins are then directly dephosphorylated by activated calcineurin, resulting in their nuclear translocation and induction of NFAT-mediated gene transcription18192+]i increase caused by the activation of TRPC1, TRPC3, TRPC4, and TRPC6 channels224123212+-dependent fashion. HF-induced apoptotic cell death was also reduced in neonatal GMCs pretreated with calcineurin/NFAT inhibitors FK506 and VIVIT. These findings indicate that NFAT is a downstream target of TRPC6 channel-dependent [Ca2+]i elevation in GMCs. The NFAT family consists of four Ca2+/calcineurin-responsive members NFATc1, NFATc2, NFATc3, and NFATc418192+]i elevation in GMCs is possible, but not demonstrated in this study.The NFAT family of transcription factors are important regulators of genes that promote differentiation, proliferation and death in a broad range of cells18192+]i chelation, calcineurin/NFAT inhibition, and TRPC6 channel knockdown. These data suggest the involvement of TRPC6-dependent calcineurin/NFAT activation in FasL and caspase-8 induction in neonatal pig GMCs. HF-induced caspase-3/7 activity was also attenuated by [Ca2+]i chelation, calcineurin/NFAT inhibition, and Fas blockade. Together, these findings indicate that TRPC6 channel-mediated [Ca2+]i elevation and subsequent activation of calcineurin/NFAT, FasL/Fas, and caspase cascades result in neonatal pig GMC apoptosis.Activation of the cell death surface receptor, Fas by its cognate ligand FasL is a major contributor to the extrinsic pathway of programmed cell death323445464547344950512+ release from the mitochondria, independently of TRPC6 channels53542+ uptake and induce mitochondrial Ca2+ release, thereby increasing [Ca2+]i562+]i which was independent of extracellular Ca2+, suggesting Ca2+ release from the intracellular stores. By contrast, HF did not stimulate Ca2+ release from the intracellular stores but induced nimodipine-insensitive and TRPC6-dependent Ca2+ influx in the cells. HF-induced NFATc1 nuclear translocation, FasL and caspase 8 activation, and apoptosis of neonatal pig GMCs were also dependent on TRPC6 channels. These data suggest that TRPC6 channels mediate the effects of HF reported in the present study. Perhaps, the molecular mechanisms underlying the pharmacological actions of HF is dependent on cell type. Also, data in this study were derived from primary neonatal GMCs. Thus, TRPC6 channel-mediated intracellular signaling may differ in adult GMCs.Studies have suggested that HF exhibit protonophore activity, and may stimulate Ca2+]i elevation and subsequent cell death may be of importance in renal dysfunctions, especially in immature kidneys.Whereas the contribution of TRPC6 channels to podocyte pathology is well known, their role in mesangial pathophysiology is poorly understood. Given the involvement of GMC apoptosis in glomerulopathy, we propose that mesangial derangement promoted by TRPC6-dependent i concentrations were measured in GMCs loaded with fura-2-acetoxymethyl ester using a ratiometric fluorescence system  as we have previously described582+ release from intracellular stores, cells were incubated in Ca2+ free modified Krebs’ solution582+ channel blocker nimodipine (1 μM).Changes in [Ca3 cells/well) were performed using CellPlayer caspase-3/7 reagent  and the IncuCyte ZOOM live content microscopy system (Essen BioScience). Cell growth and kinetic activation of caspase-3/7 were monitored in real-time and automatically acquired at two-hourly intervals by the IncuCyte interface and software.Real-time growth and kinetic quantification of apoptosis in GMCs seeded in microplates .Lipid peroxidation was evaluated in cell lysates using the TBARS kit . Superoxide generation was examined using a superoxide fluorogenic kit that specifically detects superoxide levels in live cells . The fluorescence generated by this probe was visualized and documented using a Zeiss laser-scanning confocal microscope. HGMCs were cultured in 6-well microplates as described above. Membrane FasL concentration and caspase-8 activity were quantified in cell lysates using the porcine FasL ELISA  and FLICE/caspase-8 colorimetric assay  kits. Data obtained from the cell lysates were normalized to protein concentrations.Mouse monoclonal anti-actin (Cat. No. ab3280) and rabbit polyclonal anti-TRPC6 (Cat. No. ab81111) primary antibodies and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Abcam . DMSO, hyperforin, and tert-Butyl hydroperoxide were purchased from Sigma-Aldrich. BAPTA, FK506, VIVIT, Ac-DEVD-CHO, Fas blocking antibody, ionomycin, pluronic F-127, and Fura-2 AM were purchased from Assay Biotechnology , Cell Signaling Technology , Cayman Chemical Company , Biotium Inc. , ProSpec-Tany TechnoGene Ltd , Cayman Chemical Company, Anaspec , and Life Technologies, respectively.All data are expressed as mean ± standard error of mean (SEM). Statistical significance was determined using Student’s t-tests for paired or unpaired data and analysis of variance with Bonferroni post hoc test for multiple comparisons. A P value < 0.05 was considered significant.How to cite this article: Soni, H. and Adebiyi, A. TRPC6 channel activation promotes neonatal glomerular mesangial cell apoptosis via calcineurin/NFAT and FasL/Fas signaling pathways. Sci. Rep.6, 29041; doi: 10.1038/srep29041 (2016).


CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3  mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca2+ release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca2+ handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n′,n′-tetraacetic acid (EGTA) to measure Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions.Mutations in 2+ release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student’s test with significance set at p < 0.05.Standard and confocal fluorescence microscopy was used to study Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites.We found that the peak value of Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.Overall, these results suggest that decreased Ca CAPN3 lead to limb girdle muscular dystrophy type 2A (LGMD2A), a disease characterized by progressive muscle weakness and wasting :[Ca2+]). This mixture allowed for: (a) fixing the resting myoplasmic [Ca2+]concentration in all fibers to ~80 nM , (b) recording fluorescence transients that approximate the Ca2+ release flux from the SR to the myoplasm, and (c) preventing contraction and the ensuing optical artifacts. After impalement of both electrodes, a 20-min period was allowed for equilibration between the pipette solution and the myoplasm. This assures that all measurements were performed in the presence of similar intracellular concentrations of the components of the internal solution . Calcium release was elicited by either action potentials (under current clamp conditions) or voltage pulses (under voltage clamp conditions). Action potentials were triggered by brief (0.5 ms) current pulses (15 % above threshold). Single current pulses or 1-s trains of current pulses  were used. The optical setup consisted of an inverted microscope equipped with (a) a standard epifluorescence attachment, (b) a custom-made confocal spot detection system, (c) a two-axis stage with 20 nm resolution scanning ability, (d) a CCD camera, and (e) two photodiode-based light detection systems  (resting free [Mg2+] = 0.15 mM); and (g) 15 mM Ca2+ (resting free [Ca2+] = 0.075 μM).Careported , 26 with2+] and [Mg2+]. We do not report these values since, as discussed elsewhere [2+] transients are significantly depressed with respect to those under physiological conditions. The advantage of our approach is that movement is blocked, and [Ca2+] release fluxes can be accurately estimated  concentration in this solution was estimated  to be 0.15 mM. (b) Tyrode: 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 MOPS, 10 glucose, pH = 7.4 with NaOH. (c) tetraethylammonium (TEA)-Cl: 150 TEA-OH, 10 CsCl, 2 CaCl2, 1 MgCl2, 10 MOPS, pH = 7.4 with HCl.Three solutions were used (mM): (a) internal solution: 75 aspartate, 5 ATP di-sodium, 5 phospho-creatine di-Tris, 5 reduced glutathione, 5 MgCl2O. The internal solution was used in all experiments, Tyrode was used for current clamp experiments, and TEA-Cl was used for voltage clamp experiments. For voltage clamp experiments, tetrodotoxin (TTX) (200 nM), nifedipine (20 μM), 9-anthracene carboxylic acid (200 μM), and TEA (150 mM) were used to block sodium, calcium, chloride, and potassium currents, respectively.Osmolality of solutions was 300 ± 5 mOsmol/kg Hg ×10 min × 4 °C. The following antibodies were used for Western blot analyses: anti-calpain3 12A2 (Novocastra), anti-RyR1 (Thermo), anti-vinculin (Sigma), and anti-DHPRα (Thermo).For Western blot analysis of total muscle extracts, FDB muscles were dissected and homogenized in a Dounce homogenizer in 30 volumes of reducing sample buffer  with protease inhibitors cocktail (Sigma), boiled for 2 min and centrifuged at 12,000Voltage, current, and optical data were filtered at 10, 5, and 2 kHz, respectively. Data were digitized at 30 μs/point using a National Instruments board (PCI-6221). Electrical stimulation, illumination, and data acquisition were under computer control, using a custom program written in LabView.t test. Significance was set at p < 0.05.Data are reported as mean ± SEM. Means calculated from population data were compared using the Student’s 2+ release dynamics between young C3KO and WT muscles using low affinity Ca2+ indicators that allow for study of calcium transients [2+ release in fibers equilibrated with pipette solutions containing the low affinity calcium dye OGB-5N and a high concentration of free calcium buffer . Typical electrical and fluorescence single sweep records are shown in Fig. 2+ transient, elicited by the AP shown in Fig. F/F = ΔF/F0 + A1e−t/τ1 + A2 e−t/τ2. The first (fast) exponential  accounts for ~95 % of the decay, while the second exponential  accounts for the remaining part of the transient’s decay phase. An example of the Ca2+ transient from C3KO fibers is shown in Fig. 2+ transient (ΔF/F = 0.57) is smaller than that from the WT fiber . In addition, the transients from WT and C3KO fibers reached their maximum values at similar times after the peak of the corresponding APs . As observed in Fig. 2+ transients are not due to differences in the corresponding APs, since the electrical responses differ only slightly in amplitude. The similarity in kinetics is demonstrated in the inset of Fig. 2+ signals in Fig. 2+ fluxes and shown in Fig. F/F (black traces) are shown superimposed to the experimental records  is smaller than that from the WT fiber (260 μM/ms).In the current investigation, we performed a comprehensive comparison of the Caer EGTA, 5 mM. TypF/F and Ca2+ flux amplitudes  and 265 ± 2.4 μM/ms , for C3KO and WT fibers, respectively  was statistically significant (p < 0.05).In order to determine whether the apparent difference in Δely Fig. . This re2+ release that we observed with single pulse stimulation is also present in the responses to repetitive stimulation, we recorded fluorescence transients in response to trains of pulses applied at frequencies of 10, 20, 50, and 100 Hz  in WT fibers, the same pattern was found in C3KO fibers (not shown). From this plot ; this result suggests that Ca2+ release in C3KO fibers is not sustained as efficiently as in WT fibers.In order to quantify the differences in the tetanic responses of WT and C3KO fibers, the peak (brown symbols) and inter-pulse values of every fluorescence transient along the trains (blue symbols) were approximated to envelope curves  and C3KO  were compared. In contrast, for both C3KO and WT muscle fibers, the mean amplitude of the last action potential  was not altered in C3KO, we investigated which step(s) of the E-C coupling process might be impaired in C3KO fibers. We first assessed whether the voltage dependence of the Ca2+ release was altered in the presence or absence of calpain 3. To this end, we measured fluorescence transients in response to voltage clamp pulses of various amplitudes. Since our model does not incorporate explicit equations of the voltage dependence of Ca2+ release, data are presented as ΔF/F transients. Fibers were rendered electrically passive by exchanging Tyrode by TEA-Cl. An experiment performed in a WT fiber is shown in Fig. +-dependent fluorescence increases to an early peak and then decays to a steady-state value. Both the peak (indicated by the brown dot) and the steady-state values (indicated by the green dot) depend on the pulse amplitude, and both seem to saturate for large voltage pulses. Finally, the signals slowly relax to a baseline after the pulses end. To quantify the voltage dependence of the Ca2+ release, the peak and steady-state ΔF/F values of every transient were plotted as a function of the membrane potential, and the data were fitted to a Boltzmann function  and had slopes of 13 and 11 mV (k) for the peak and steady-state values, respectively. A similar experiment for a C3KO fiber is shown in Fig. F/F transients in the C3KO fiber has a similar profile as that of a WT fiber, the corresponding values for V1/2 (3 vs. 0 mV) and k (12 vs. 10 mV) values are smaller for C3KO.Since the physiological trigger for Caion Fig. . In thisF/F values in this example are 0.6 and 0.28, representing ~22 and ~25 % reductions in C3KO with respect to the WT values. The population analysis for the voltage dependence of Ca2+ release is summarized in Fig. F/F transients in populations of WT (n = 10) and C3KO (n = 20) fibers, each from two mice. The maximal peak ΔF/F for C3KO was 20 % smaller than those from WT fibers, while the maximal steady ΔF/F was reduced by 25 % in C3KO fibers. The statistical significance of such differences is shown in Fig. V1/2 and k calculated from data acquired from WT and C3KO mice. The inset in Fig. V1/2 and k values. In addition, data in Table V1/2 values from WT and C3KO fibers.The peak and steady-state Δf WT n = 0 and C3Kk and V1/2 parameters used to fit the voltage dependence of Ca2+ release from WT fibers (and C3KO fibers) reported here are in good agreement with those previously reported by us and others for FDB and interosseous fibers [The s fibers , 27–29.2+ release; thus, we assessed the expression of both DHPR and RyR1 in FDB muscles from young mice used in the current investigation  of the voltage-dependent release sites among the triads.While the biochemical data presented here are consistent with the above Ca2+ release in C3KO fibers. Representative results of one fiber from each strain are shown in Fig. 2+-dependent fluorescence changes recorded in a WT fiber. Six positions where the fluorescence reaches maximal values can be clearly identified; those sites correspond to the location of triads [To assess the topology of the predicted reduction in release sites  per triad, we performed a confocal study of AP evoked Caf triads , 25.Fig.2+ diffusion away from these sites leads to the dynamic formation of spatiotemporal Ca2+ microdomains; they are centered at each transverse tubule (triad) and separated from each other by minima at the Z and M lines, the later indicated by arrows [2+ release process. To provide a notion of the spatiotemporal formation of the Ca2+ microdomains, we present the same data in Fig. 2+ release is also stereotypically observed along the ~3 sarcomeres scanned, with the deepest valleys at the M lines (indicated by arrows) and maxima at the triads. Furthermore, consistent with the results using global illumination/detection described above, the peak amplitudes of the Ca2+ microdomains in the C3KO fiber are smaller than those in the WT fiber; notably, all the peaks are similarly reduced. Comparing the contour maps in Fig. 2+ release in C3KO and WT fibers, we studied the Ca2+ release from six WT and six C3KO fibers, obtained from two mice of each strain, and compared the ΔF/F transients and Ca2+ flux transients recorded at relevant positions along the sarcomere , the Z line (Z), and the M line (M)). To this end, we averaged the records at each position from 10 μm scans from each fiber and subsequently computed the mean values from all fibers of each strain  is significantly larger than the 220 ± 7.4 μM/ms in C3KO fibers. Overall, these calculations demonstrate the Ca2+ release fluxes calculated at the triads are larger than those calculated from global recordings, which are space-averaged.We finally used the fluorescence transients occurring at T locations (triads) to calculate the Cacroscope ). The av2+ release in a mouse model of LGMD2A and conclude that the absence of CAPN3 leads to impaired Ca2+ handling as an early feature of disease. We combined electrophysiological and optical methods to measure Ca2+-dependent fluorescence changes (ΔF/F transients) in isolated FDB fibers, loaded with a low affinity Ca2+ dye and high EGTA concentrations and maintained in current or voltage clamp conditions. Ca2+ fluxes were calculated from ΔF/F transients recorded using space-averaged  and space-resolved  detection techniques.In the current study, we conducted a detailed analysis of Ca2+ measurements were performed in aged mice, we were concerned that the defects in Ca2+ release might have been a consequence of structural abnormalities that accumulated over time. The data generated here showed that Ca2+ release was reduced and the expression level of both RyR and αDHPR were decreased in young C3KO mice compared to age-matched WT mice. Thus, these changes appear to be largely independent of the degenerative processes that occur in the pathogenesis of C3KO muscles during disease progression.We conducted the studies here on young mice because the phenotype of C3KO mice is progressive as the mice age . Since o2+ fluxes elicited by single action potentials in C3KO fibers are significantly smaller than those from age-matched WT mice. These changes in amplitude are not accompanied by alterations in the kinetics of the flux transients. The limitation seen in Ca2+ release in response to single stimulation is more pronounced during repetitive stimulation, which is the physiological pattern used by the CNS to recruit mechanical output of muscles. Also, this attenuation in Ca2+ release increases with frequency, and this characteristic is expected to impact muscle mechanical output and fatigue resistance. A detailed comparative analysis of the electrophysiological data  in response to voltage pulses. The slope and the midpoint voltage for activation of Ca2+ release from WT and C3KO are indistinguishable from each other. Instead, only a depression of both the peak and steady-state Ca2+ fluxes was found. Both values were reduced by ~20 % with respect to control values; a depression similar to that found in AP evoked Ca2+ fluxes. These results demonstrate that the voltage-sensing step of the E-C coupling process, i.e., the response of the αDHPR to membrane depolarization, is not altered in C3KO, while Ca2+ release is scaled down. These findings point towards possible alterations in the gain of the transduction process or the release process itself as the cause of the impaired Ca2+ handling.We found no voltage dependence alteration in either the peak or steady-state of Δ2+-dependent fluorescence changes [2+ release along the sarcomeres of C3KO with no sign of altered or failing triads along the stretches scanned  release sites per triad is uniformly reduced along the sarcomeres.Although it is not clear why the concentrations of both DHPR and the RyR1 are significantly reduced in C3KO mice Fig. , our bio changes , 25, we ed Figs.  and 10. One explanation for why the absence of CAPN3 leads to reductions of both DHPR and RyR1 may be related to the fact that the total amount of DHPR is tightly regulated in adult fibers  and is r2+ fluxes (14–20 %) and the levels of both αDHPR (40 %) and RyR (60 %) is intriguing. In principle, the large reduction in αDHPR and RyR1 predicts a larger reduction in Ca2+ fluxes. Possible explanations are that not all the αDHPR and RyR1 participate in Ca2+ release in WT animals or alternatively that the gain of Ca2+ release is in fact increased in C3KO mice, thus partially compensating for the reduced number of release sites. Another possibility is that the impaired Ca2+ release at the triads of C3KO mice leads to a reduced Ca2+-dependent inactivation of the RyR, which partially compensates for the downregulation of RyR expression. Moreover, it is possible that the content of RyR1 and αDHPR is dependent on fiber diameter . Since the C3KO electrophysiological data were obtained from a population of relatively large (and uniform) diameter fibers, it is possible that their content of RyR1 and αDHPR is not as low as expected from biochemical data.The disparity between the reductions of Ca2+ in skeletal muscle can affect several downstream signaling pathways that link muscle contractile activity with gene expression. This link is extremely important as it allows muscle to adapt as needed to the level and type of activity necessary, for example, by gradual transition of muscle fiber phenotype  or by switching the metabolic profile . One such signaling pathway is mediated by CaMKII, which activates the transcription factor MEF2, to enhance gene expression of slow-type genes and facilitate the transition from fast to slow fiber phenotype. CaMKII signaling and several aspects of muscle adaptation are blunted in C3KO muscles [In prior studies, we and others , 8 demon muscles  (and dat2+ release may be an early defect in the pathogenic cascade of LGMD2A and that reduced Ca2+ release precedes impaired CaMKII signaling. The results are important because they establish key features of the pathogenic cascade in LGMD2A. Future studies are necessary to elucidate the precise role of CAPN3 at triads and its role in Ca2+ handling in skeletal muscle.These results suggest that decreased Ca


Mary Ellen Avery’s research is recognized as a milestone in biomedical research. She had discovered the underlying cause of hyaline membrane disease, surfactant deficiency, fostering ever more vigorous efforts to reduce neonatal mortality in the burgeoning practice of Neonatology. Neonatology is the only clinical discipline that began as an experiment, making it a model for biomedical research. Avery knew that the concerted effort to treat preterm newborns could potentially do more harm than good, violating her oath to Hippocrates, if not held to the highest scientific standards. She remained true to that pledge throughout her career, as recounted in this Review. I feel like the narrator recounting his tale at the wedding banquet in Coleridge’s epic poem “The Rime of the Ancient Mariner.” Of course anyone can go to PubMed and retrieve Mary Ellen Avery’s publications, totaling 146 peer-reviewed papers, but the back story is what I am going to relate, largely based on my personal recall under her tutelage for 25 years.Her interest in the breathing problems of newborn infants had been piqued by the awareness that the most common finding in the lungs of premature infants born alive who died shortly thereafter, was atelectasis and hyaline membranes. The pathology had been well-described by both George Anderson and Peter Gruenwald at Johns Hopkins. They both emphasized the lack of a clinical description of the course of the disease. In 1947, Gruenwald .The Lung and its Disorders in the Newborn Infant (There were multiple theories for the pathogenesis of HMD when Avery began her study of lung surfactant. In the first edition of her book n Infant , she objThe first four peer-reviewed papers Avery published were case reports, beginning in 1955 . But thein utero came when Liggins discovered that antenatal glucocorticoids could accelerate lung maturation. Avery’s laboratory performed a systematic series of studies to demonstrate the physiologic effect of glucocorticoids on lung surfactant production in both rabbits (The breakthrough in the treatment of surfactant deficiency  rabbits –23 and l rabbits , includi rabbits  for “ful rabbits , 27 so a rabbits  showing  rabbits , capping rabbits .In 1984, on the 25th anniversary of the publication of the Avery and Mead paper, members of the Joint Program in Neonatology, which Dr. Avery had created in 1974, gathered for a group picture with her in front of the Administration Building at Harvard Medical School (see Figure In addition to her research efforts, Dr. Avery was a champion for women in the field of medicine. Along with Lynn Reid and Mary Ellen Wohl, she strongly advocated for a level playing field as the first woman ever to have become the Chair of a clinical department at Harvard Medical School. When she was applying to medical school she was rejected by Harvard Medical School because of their policy of excluding women – always the student of history, fighting the hard fight for what was right despite the prevailing attitudes, whether in science or social justice.Dr. Avery’s last cited paper, entitled “What is good for children is good for mankind: the role of imagination in discovery” was her formal Address to the American Association for the Advancement of Science as the President of the society in the year 2004 . In her I can only hope that her spirit will marshal on.The author declares 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 two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca Intercellular signalling can be mediated by gap junction channels, and calcium blocks this signally during tissue injury. Here, the authors use X-ray crystallography and molecular dynamics to show that the calcium forms an electrostatic barrier to prevent transport of cations. Connexins (Cx) are a 21-member family of integral membrane proteins with four transmembrane (TM) domains M1–M4), two extracellular loops (E1 and E2), a cytoplasmic (CL) M2–M3 loop and cytoplasmic N- and C-terminal domains , with a molecular weight (MW) cutoff of about 1,000 Da and a size limit of up to ∼14 Å (refs 8Vj)182+ H32 space group with two monomers in the asymmetric unit ; ; ref.  resulted3); ref. . MR withabc=75 × 75 × 40 Å3; 2+-bound Cx26 structure was built and ultimately refined to 3.3 Å resolution (R/Rfree=0.280/0.313). Although the maps are generally of high quality for the TM α-helices and the extracellular loops, the NT, CL and CT domains could not be modelled because the electron density in those regions is not well defined. The final model consists of residues 19–95 and 134–216 for protomer A and residues 21–95 and 134–213 for protomer B. The Ca2+-bound Cx26 structure was used as a search model for MR to solve the structure of Ca2+-free Cx26 at 3.8 Å resolution  and multi-angle light scattering indicate that Cx26 purified in FA-3 is a dodecamer , formingAlthough the maximum RMSDs between our structures and 2ZW3 are substantial , our ind2+, Fo–Fc difference density maps display two discrete 5 σ peaks in the AU between subunits at the M1/E1 boundary , the coordination number (CN=5) and the formal charge of the site (-2) are consistent with mean values reported in an analysis of nearly 1,500 non-EF hand Ca2+-binding proteins for which high-resolution crystal structures were availableIn the presence of Cagreen in , corresp2+-bound and Ca2+-free electron density maps corroborates the identity of the Ca2+ ions: the peaks ascribed to Ca2+ in the Ca2+-bound Cx26 structure were absent in the Ca2+-free Fo–Fc maps (2+ ions is therefore supported by (i) the geometries and distances to coordinating oxygen atoms, (ii) the Fo–Fc  anomalous difference maps from the Ca2+-bound crystals 2+ ions in adjacent binding sites are separated by 16 Å, with a 60° rotation around the pore axis. The maximum distance between the Ca2+-binding sites across the pore lumen is 23 Å. The Ca2+-binding sites coincide with the region of closest approach between the pore-lining M1 helices of adjacent subunits in the hemichannel. However, the major inter-subunit interface is between M1 and M2 (> 500 Å2), and includes a salt bridge between E42 and R75, which is not present in the Ca2+-free structure, as discussed below. The proximity of the adjacent M1 helices in this region and the substantial M1–M2 contact surface are both conserved in the presence or absence of Ca2+ binding  simulations discussed below). The carboxylates of the glutamate residues act as coordinating bidentate ligands, with equivalent Ca2+ coordination distances and angles between the Oɛ1 and Oɛ2 oxygens.Ca subunit . Thus, iN=5 ref. . Hemisph channel , where t2+-bound and Ca2+-free channel structures are nearly identical, with a limiting diameter of ∼15 Å  to form an intrasubunit salt bridge with K188, the position of which is essentially invariant between the two structures  lipid bilayers. Each system included explicit water molecules and was neutralized and ionized with 140 mM KCl in the cytoplasmic region and 140 mM NaCl in the extracellular region  followed by a production phase  was stable  for K+ and Cl− along the Ca2+-bound and Ca2+-free channel axes based on ion density distributions in the all-atom simulations. The resultant K+ and Cl− free energy profiles  insect cells with recombinant baculovirus. Cells were infected at a multiplicity of infection (MOI) of 5 and harvested 48–52 h post infection. All subsequent steps were performed at 4 °C, unless otherwise indicated. Cells were lysed by 20–25 strokes using a Dounce homogenizer in low-salt buffer ). Nucleic acids were digested by adding 10 units of Benzonase (EMD Biosciences), adjusting the buffer to include 2.5 mM MgCl2, followed by 15 min of gentle rocking. The lysate was clarified by ultracentrifugation , and the pellet was washed with low-salt buffer, high-salt buffer  and final wash buffer . The final pellet contained enriched plasma and organellar membranes. Membranes were stored as aliquots, flash frozen in liquid N2 and stored at −80 °C until use.Expression of human Cx26 C211S, C218S (in which two cysteine residues in the CT were replaced with serine to prevent non-native disulfide bond formation) with a C-terminal hexahistidine tag was performed by infection of N-decyl-β-D-maltopyranoside (DM) detergent (Anatrace) was added to the resuspended membranes to a final concentration of 1% ). Membranes were solubilized for 3 h on a rotator and then clarified by ultracentrifugation . After detergent solubilization, it was found that maintaining the protein in buffers with high ionic strength (0.5–1.0 M NaCl) was essential to ensure protein stability and solubility during affinity purification and subsequent concentration steps. His-tagged Cx26 was purified from solubilized membranes by binding the protein to immobilized cobalt affinity resin (Clontech) at a ratio of 1 ml affinity resin per 50 ml detergent extract. After batch binding for 30–60 min, the slurry was poured into a column, and the beads were washed with at least 5 column volumes (CVs) of extraction buffer with 1% (w/v) DM, followed by 5 CVs of wash buffer  DM, 20 mM imidazole and 1 × Protease Inhibitor Set V). DM was exchanged for FA-3 on the column by addition of two CVs of exchange buffer  glycerol and 1 × Protease Inhibitor Set V) containing 0.08% (w/v) FA-3 detergent (4 × CMC) to the resin, and 1 CV was allowed to flow through the column. The column was capped, kept stationary and incubated with the new detergent for 15 min. The resin was further washed with 1 CV of FA-3 exchange buffer. Cx26 was then eluted from the column in 1 ml fractions with elution buffer (exchange buffer containing 250 mM imidazole) containing 0.05% FA-3. Fractions with the highest protein concentrations, as estimated by absorption at A280 , were pooled and concentrated to ∼0.5 ml in a 100-kDa molecular weight cutoff (MWCO) concentrator (Millipore). Imidazole was removed using a G-25 desalting column  equilibrated in final buffer  glycerol and 1 × Protease Inhibitor Set V) containing 0.02% (w/v) FA-3. For crystallization trials, the eluate from the desalting column (∼1 ml) was concentrated to 4–6 mg ml−1 (150–200 μM) using a 500-μl or 4-ml concentrator .Membranes (∼5 g wet weight) were thawed on ice and resuspended in 50 ml extraction buffer  glycerol and 1 × Protease Inhibitor Set V ). Membranes were disrupted by ten strokes using a Dounce homogenizer in order to obtain a small particle size for efficient solubilization. A 10% (w/v) stock solution of 2 and 12% PEG 3350. Crystals for X-ray diffraction experiments were on average 50 × 50 × 25 μm3 and typically grew within 1–2 weeks in the presence of 5–50 mM CaCl2. The best diffracting Ca2+-bound crystals were grown from setups with 20 mM CaCl2. To grow Ca2+-free Cx26 crystals, the same crystallization setup and reservoir buffer were used, with the CaCl2 omitted from the formulation. In the absence of CaCl2, crystals required at least a month to grow. Cryoprotectant (either glycerol or ethylene glycol) in harvesting buffer , 14% PEG3350 and 0.02% FA-3) was added directly to the crystallization drop by increasing the concentration of glycerol or ethylene glycol in the crystallization drop in 5% increments every 5–10 min. Crystals were then harvested directly from the drop into 50–100 μm diameter loops (Hampton Research), flash frozen by plunging into liquid N2 and loaded into ALS-style pucks.Crystals of Cx26 in FA-3 could be grown reproducibly at 25 °C using hanging-drop or sitting-drop vapour diffusion methods by mixing 1 ml of Cx26 in final buffer with 0.02% FA-3 and 1 μl of a reservoir composed of 100 mM HEPES, pH 7, 15 mM sodium formate, 20 mM CaCl2+-bound Cx26 crystals, X-ray diffraction data were collected at the SER-CAT 22-ID beam line at the Advanced Photon Source at Argonne National Laboratory. Initial indexing of the crystals by HKL2000 (ref. H3), a maximal diffraction resolution of 3.0 Å and a mosaicity of 0.5°. Using a 50-μm beam and 1° oscillations, several wedges of data were collected from one crystal. Ultimately, three data sets were integrated separately and merged upon scaling, with an overall Rmerge of 7.5% and a resolution cutoff of 3.1 Å.For the Ca000 ref.  suggeste2+-free Cx26 crystals, X-ray diffraction data were collected at the GM/CA-CAT 23-ID beam line at the Advanced Photon Source. The Ca2+-free crystals had the same Bravais lattice and were isomorphous with the Ca2+-bound crystals, although reflections could only be observed to 3.6 Å resolution. Two wedges of data from one crystal were merged upon scaling, with an overall Rmerge of 7.5% and a resolution cutoff of 3.8 Å. Both crystal forms (Ca2+-bound and Ca2+-free) conformed to the H32 space group with two molecules in the AU with a Matthews coefficient (Vm) of 3.7 and a solvent content of 67%. Crystallographic statistics are listed in 1/2 2 resolution cryoEM map derived by analysis of two-dimensional crystals of Cx43  was coincident with the crystallographic twofold. We converted the Cα model to polyalanine (polyAla) in CootWe solved the Cax43 ref. . These tσ. The final monomeric model based on the cryoEM map consisted of 184 residues. We generated a dimer of the expanded model by application of the NCS operators determined from the dimeric 4-TM polyalanine model that we built with the aid of the initial X-ray crystallographic maps. It was important to use the NCS operators based on the X-ray crystallographic map to generate the dimer because the relative orientations of the Cx subunits differed slightly between the two- and three-dimensional crystals. The expanded dimeric model consisted of 368 residues.We then used the cryoEM mapH32 X-ray crystallographic data as the target. The search included data from 35 to 3.5 Å resolution and succeeded with a top translation function Z-score of 6.9. Rigid body refinement of the MR model in Phenix yielded crystallographic R and Rfree values of 0.511 and 0.526, respectively, and 2Fo–Fc maps clearly showed density for the full dodecameric channel  and the carboxylates of a second glutamate (E47) and the carbonyl oxygen of a glycine (G45) from the adjacent Cx subunit  and calculated an anomalous difference map at 4 Å resolution using Ca2+-free, model phases. The only peak observable at >4-σ in the anomalous map partially overlapped with one of the Fo–Fc difference density peaks (+5-σ) that had guided the placement of a Ca2+ atom , formal charge of the site (n=−2) and coordination bonding distances (both average side chain carboxylate distance and average main chain carbonyl distance=2.6 Å) were all within the reported ranges for Ca2+ binding. Although the resolution limit of the diffraction data precluded the definitive placement of water molecules, it is suspected that the residual positive density was likely due to the presence of coordinating water molecules. Water coordination is expected given the hemispheric and incomplete coordination of the Ca2+ by the protein. MD simulations of the Ca2+-bound channel also showed hydration of the bound Ca2+ ions , prime-and-switch NCS averaging and density modification resulted in maps that allowed us to obtain a well-refined model of the Ca2+-free GJC. Significantly, no peaks in either Fo–Fc or anomalous maps appeared in the vicinity of the Ca2+-binding site identified in the Ca2+-bound structure.We solved the Ca2+-free channel, we used the same method to add intracellular CaCl2, in order to make the total Ca2+ equivalent in the simulations. (No cytoplasmic Ca2+ was added to the simulations of the Ca2+-bound channel.)We used VMD 1.9.1 ref.  to buildWe used the CUDA-enabled version of NAMD 2.9 to perform the equilibrium MD simulations of these systems using the CHARMM36 force field for the POPC bilayerswww.ks.uiuc.edu/Training/Tutorials/science/membrane/mem-tutorial.pdf. We first equilibrated the lipid acyl chains, keeping the rest of the system fixed. Next, we restrained the protein in order to first minimize and equilibrate the rest of the atoms of the system. Then, we released the restraints on the protein and equilibrated the entire system. Finally, for all-atom production runs employing Langevin dynamics, we used a constant temperature of 310 K and constant pressure of 1 atm with 2 fs time steps.For each simulation, minimization and equilibration followed the procedure described at http:// 2+ interaction energy is underestimated in the CHARMM27 force field2+-bound simulation with the G45O-Ca2+ distance restrained to 2.35 Å, with a spring constant of 40 kcal mol−1.The carbonyl oxygen–Ca+ and Cl− ion density distributions along the Ca2+-bound and Ca2+-free Cx26 GJC pore (the Z axes) during 30 ns of the all-atom MD simulation production phases (+ or Cl−) were counted at each position along the Z axis with a fixed radius equal to the minimum channel radius  and a ΔZ of 5 Å. Ion densities (<ρ(Z)>) were calculated by dividing the total number of ions at each position on Z axis by the corresponding cylindrical volume. PMFs were ultimately calculated by solving the equation,PMF values were calculated based on the Kn phases . Ions  methodɛ (r)∇φ (r))=ρ (r)), where ɛ is the dielectric constant, φ is the electric potential and ρ is the surface charge density. REBEL uses the boundary element method to solve the Poisson equation for the model system described above, where the boundary is the triangulated analytical molecular surface as described by ConnollyElectrostatic surface potentials of the CaThe parameters for the REBEL boundary element electrostatic calculations were as follows. Partial charges for atoms of amino acids were assigned according to the molecular mechanics force-field named Empirical Conformational Energy Program for Peptides (ECEPP)2+-bound Cx26, we performed REBEL calculations with and without the explicit inclusion of the waters that were found to complete Ca2+-coordination in the MD simulations, and we found no significant difference in the resultant electrostatic surface potentials.In the case of CaElectrostatic surface potentials were also calculated using Poisson–Boltzmann equations as implemented in Adaptive Poisson-Boltzmann Solver (APBS). Surfaces calculated in this way are essentially identical to surfaces calculated with the REBEL method implemented in ICM-Pro.http://weblogo.berkeley.edu) to generate the diagram shown in Homologous sequences from residues 40–50 (Cx26 numbering) for all 21 human Cx isoforms were defined by a sequence alignment performed with PRALINEAccession codes: Coordinates and structure factor files for Ca2+-bound and Ca2+-free Cx26 have been deposited in with the Protein Data Bank under PDB ID 5ER7 and 5ERA, respectively.How to cite this article: Bennett, B. C. et al. An electrostatic mechanism for Ca2+-mediated regulation of gap junction channels. Nat. Commun. 7:8770 doi: 10.1038/ncomms9770 (2016).Supplementary Figures 1-10, Supplementary Tables 1-2, Supplementary Discussion and Supplementary ReferencesCa2+ coordination sites reside at the interface between adjacent subunits, near the entrance to the extracellular gap, where local conformational rearrangements enable Ca2+-chelation. Each of the 12 Ca2+ binding sites is comprised of the G45-O and the E47 carboxylate from one subunit, and the E42 carboxylate of the adjacent subunit. The Ca2+-bound and Ca2+-free structures were superimposed, and the morph conformations tool in Chimera was used to create a conformational trajectory between the two structures .A view showing the transition between the Ca2+-bound and Ca2+-free gap junction channels, highlighting the E47:K188 salt bridge in the Ca2+-free structure. Molecular dynamics (MD) simulation of a Ca2+-bound gap junction channel shows that the Ca2+ ions induce a strongly positive electrostatic potential that dramatically restricts K+ permeation. Six Ca2+ ions (yellow) bind at the interface between adjacent subunit within the hemichannels that dock end-to-end to form a gap junction channel. All atom MD simulations were performed on the Cx26 X-ray structure (cyan) embedded in two POPC bilayers . The cytoplasm and extracellular spaces were ionized with 140 mM KCl  and 140 mM NaCl , respectively.  Water molecules are shown as blue lines.Ca2+ ions remain stably bound throughout the MD simulation, with minimal movement of the protein backbone. The E47 carboxylate from one subunit and the E42 carboxylate of the adjacent subunit are stably coordinated throughout the MD simulation, as are three water molecules (red and white sticks). However, coordination by G45-O was only maintained at 1 out of 12 sites in the unrestrained simulation. The Ca2+ coordination geometry determined by X-ray crystallography is maintained throughout the MD simulation at one of the binding sites. Coordination by G45-O was maintained at this particular site. Restrained MD simulations also maintained coordination by G45-O at every binding site. The electrostatic properties of the channel were not influenced in either the restrained or unrestrained MD simulations. Stick representations and colors are as in MD simulation of the Ca2+-free gap junction channel shows that the channel is highly permeable to K+ ions. During the MD simulation, K+ and Cl- ions enter and traverse the channel, and K+ ions cluster in the vicinity of anionic residues at the Ca2+ binding sites.