Nevertheless, some viroporins are known to disrupt host cell Ca2+ homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca2+ in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca2+. Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined “square top” openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca2+ and Ba2+) as monovalent cations (K+), but a viroporin defective mutant lacked Ca2+ conductivity. These data demonstrate that the NSP4 VPD is a Ca2+-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca2+ homeostasis.Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca However, a small number of viroporins are retained in the ER membrane and significantly deplete ER Ca2+ levels, suggesting that these viroporins target ER Ca2+ stores. Rotavirus (RV) NSP4 and picornavirus 2B proteins are the two best-characterized viroporins for the disruption of cellular Ca2+ homeostasisIon channels are part of the fundamental cellular machinery used by cells to transduce signals across cellular membranes. As obligate cellular parasites, viruses too express ion channels to subvert cellular conditions in favor of virus replication and contribute to pathogenesis. Virus-encoded ion channels are called viroporins. Thus far, viroporins have been predominantly identified in animal viruses and are characterized as monovalent cation selective channels, with varying degrees of selectivity of potassium (K2+ ([Ca2+]c) resulting from an increase in Ca2+ leak from the ER and increased Ca2+ influx through the plasma membrane (PM). Elevated [Ca2+]c is required for multiple processes of RV replication, including activation of autophagy, nucleation of RV replication complexes called viroplasms, and assembly of the outer capsid protein VP7672+ activates an ER Ca2+ sensor protein called stromal interaction molecule 1 (STIM1) and STIM1 in turn induces Ca2+ influx through store-operated calcium entry (SOCE), a homeostatic cellular mechanism to maintain ER Ca2+ levels. We further found that NSP4 viroporin activity is sufficient to activate STIM1 but NSP4 mutants that are deficient in viroporin activity neither activate STIM1 nor induce Ca2+ influx through SOCE2+, these data confirmed that NSP4 viroporin activity is responsible for the loss of ER Ca2+ in RV-infected cells and is the trigger for elevating [Ca2+]c, which is critical for RV replication.RV is one of the leading causes of viral gastroenteritis in children, and despite multiple effective vaccines, RV kills 215,000 children under 5 years of age each year2+, implies that the NSP4 viroporin domain forms a pore or ion channel that can conduct Ca2+. The purpose of this study was to first demonstrate that the NSP4 viroporin domain is able to form a bona fide ion channel and to determine whether NSP4 viroporin domain ion channels can conduct Ca2+ ions. Using both lipid bilayer and patch clamp electrophysiology techniques, we have confirmed that NSP4 is a cation-selective ion channel that can conduct both monovalent and divalent cations, including Ca2+ and Ba2+.Our previously proposed model, in which NSP4 viroporin activity depletes ER CaThe NSP4 short peptide construct encompassing the viroporin domain  was designed  and syntsn-glycero-2-phosphocholine Type II-S) was purchased from Sigma . All salts , Hepes, MOPS, K-EDTA, and sucrose were also obtained from Sigma with at least >99.0% purity. Hexadecane was obtained at TCI  and pentane (high purity solvent grade) from Burdik & Jackson . DMSO was obtained from EMD Millipore . For giant unilamellar liposomes, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and cholesterol were purchased from Avanti Polar Lipids .Asolectin  where the two halves are separated by a 0.01 mm-thick polytetrafluoroethylene  film (Goodfellows). Within the center of the Teflon film, an aperture of approximately 100 μm in diameter was created utilizing a High-Frequency Spark Tester PPM MK3 (Buckleys Ltd.) adjusted to 45 kV. In order to create a hydrophobic interface to attract lipids, the aperture was treated with a 1% hexadecane in pentane solution. One and a half mL of buffer T  or PA  was added to each half of the chamber. Five microliters of asolectin dissolved in pentane at a concentration of 5 mg/mL was added to the surface of the buffer on both sides. Ag/AgCl electrodes were then inserted into the buffer solutions. The chamber-electrode configuration was then allowed to equilibrate before formation of a lipid bilayer over the aperture. A membrane bilayer was formed over the aperture by lowering and raising the buffer level in one of the half-chambers and monitoring the resistance on the oscilloscope. A high resistance indicated that a bilayer had formed. A transmembrane voltage of + or −90 mV was then applied to check membrane stability for 10 minutes. This was also performed to ensure that there was no contaminating channel-like activity prior to the addition of channel-forming protein. If the stability test showed no contaminants and a stable baseline, then NSP4 peptide dissolved in DMSO or water (4.0–8.0 μg) was added to one half-chamber, defined as the A dehydration-rehydration method was used to reconstitute NSP4-VPD into asolectin liposomes as describedAn alternative technique for liposome reconstitution (hereafter referred to as “the cloud method”) was also performed2, 0.1 mM EDTA, 5 mM Hepes, pH 7.2) or buffer BA . Reconstituted proteoliposomes were incubated in buffer B (buffer A + 20 mM MgCl2) to induce swelling of unilamellar blisters from the liposomes for patch-clamp experiments. The pipette was immersed in the bath with constant positive pressure and micro-manipulated to contact the blister to achieve 1.0–10.0 GΩ seal. Patches were excised with brief exposure to the air and then re-immersed in the bath solution. This solution was then quickly exchanged for either buffer A, buffer BA, or buffer CA . All potential offsets were canceled electronically in symmetric buffer A before data acquisition. The reference electrode (World Precision Instruments) was in contact with the bath solution via a 3 M KCl agar bridge. Control experiments performed on liposomes lacking reconstituted NSP4 showed no channel activity.The pore-forming activity of NSP4 peptide was investigated with the traditional patch-clamp technique, as describedtrans side in planar lipid bilayer, and the bath in patch-clamp). Currents were recorded with an Axopatch-1D amplifier (Axon Instruments) and a CV-4 or a CV-4B head-stage in the patch-clamp or planar lipid bilayer setup, respectively. For recordings up to 10 minutes, currents were filtered at 500 Hz through a low-pass Bessel filter and digitized at 1.25-ms sampling intervals . For shorter high-resolution recordings of 1–2 minutes, the currents were filtered at 1 kHz and sampled at 100 μs sampling intervals. Currents were digitized using the Acquire software (Bruxton), and analyzed with pClamp (Axon Instruments).Experiments with either the planar lipid bilayer or the traditional patch-clamp technique were performed under voltage-clamp  were generated by the electro-swelling method using the Vesicle Prep Pro machine  according to the manufacturer’s instructions. GUVs were generated by applying 20 μL of 10 mM DPhPC + 10% cholesterol dissolved in chloroform onto the slide, allowed to air dry, and 1 M sorbitol was used for the external solution for GUV swelling. GUVs were collected and either left as protein-free GUVs (negative control) or reconstituted with 10 ng/mL of the NSP4-VPD peptide dissolved in water for 1 hr at room temperature. After reconstitution the GUVs were kept at 4 °C until use. For patch clamp of GUVs we used the Port-a-Patch system (Nanion) with 8–12 megaohm patching chips (~1 μm aperture). Briefly, patches were established in symmetric 650 mM KCl buffer by adding 5 μL of protein-free or NSP4-reconstituted GUVs to the chips under slight negative pressure (−10 to −40 mbar). Upon contact with the aperture, the GUV ruptures to form a bilayer with high seal resistance (>10 Gigaohm) that was very reproducible. Once seals were established, residual sorbitol was removed by exchanging the bath buffer 3 times with 20 μL of 650 mM KCl and potential offsets were canceled electronically before data acquisition. Relative selectivity between cations and anions was performed by running voltage steps from −100 mV to +100 mV initially in symmetric 650 mM KCl and then after bath buffer exchange with 65 mM KCl to generate a 1:10 asymmetric 650 mM KCl/65 mM KCl gradient across the bilayer. Patch clamp data were acquired using an EPC10 amplifier and PatchMaster software . The measured open channel current for each voltage was plotted and a linear regression line fitted to determine the reversal potential. The expected KE. coli lysis assay was performed as previously describedE. coli bearing expression plasmids for wild-type NSP4 VPD (VPD-WT) or the viroporin defective mutant (VPD-Mut) were grown overnight. The NSP4 VPD-WT and VPD-Mut constructs included a C-terminal StrepII tag for immunoblot analysis. Overnight cultures were diluted 1:100 into fresh LB media supplemented with 1% glucose, 100 μg/mL ampicillin, 35 μg/mL chloramphenicol, and grown until the optical density at 630 nm (OD630) was 0.4 to 0.6 (approximately 3 hrs). Then 1 mM IPTG was added to induce NSP4 expression or left uninduced as a negative control. OD630 measurements of each culture were taken before IPTG induction and at 10 min intervals after induction for 90 min. Additional samples were taken at 0, 10, 30, 60 and 90 min post-induction for immunoblot analysis as previously described630, and for samples of cells that had lysed, the OD630 just prior to lysis was used for normalization. Antibodies used were mouse monoclonal anti-StrepII antibody and alkaline phosphatase-conjugated goat anti-mouse IgG were used for detection.The viroporin-mediated 630 of 0.5–0.6, protein expression was induced with 1 mM IPTG and cultured for 90 min. Cells were pelleted by centrifugation  and resuspended in 2.5 mL PBS . The sample was then sonicated in PBS using a probe sonicator with two 1 min intervals. Membranes were pelleted by centrifugation  and the supernatant was collected as the soluble fraction [S]. The membranes were resuspended in ice cold 100 mM sodium carbonate, incubated for 30 min on ice, and pelleted again by centrifugation . The supernatant was collected as the peripherial membrane protein fraction [P] and the pellet was solubilized in 1% SDS in PBS for the integral membrane protein fraction [I]. Equal buffer volumes were used to maintain the same relative protein concentration as that of the starting material. Equivalent amounts of each fraction were analyzed by SDS-PAGE, as above. Antibodies used were rabbit anti-NSP4(120–147) peptide antiseraNSP4 membrane fractionation was performed similarly to our previous NSP4 viroporin study-NSP4120–7 peptide2 buffer. Aliquots of this final liposome solution were loaded onto a 96-well plate and a time course of fluorescent signals (494/518) were measured using a FlexStation 3 (Molecular Devices) before and after the addition of either 15 μM VPD-WT, 15 μM VPD-Mut, or vehicle (water).The defined lipids 1-palmitol-2-oleoyl-sn-glycerol-3-phosphoethanolamine, 1-palmitol-2-oleoyl-sn-glycerol-3-[phosphor-rac-(1-glycerol)], and 1-palmitol-2-oleoyl-sn-glycerol-3-phosphocholine were solubilized in chloroform and mixed in a 3:1:1 weight-ratio respectively and the solvent was then evaporated using argon gas. Dried 1 mg lipid-cakes were hydrated in 0.5 mL 25 mM Hepes pH 8, 150 mM NaCl, and 50 μM Fluo-5N pentapotassium salt buffer. The lipid suspension was then extruded through a polycarbonate membrane with 1 μM diameter pores. Fluo-5N that was not trapped inside the liposomes was removed by running the solution through a 3.5 mL PD midiTrap G-25 column equilibrated with a 25 mM Hepes pH 8, 150 mM NaCl buffer. Fractions were collected and tested for free Fluo-5N dye and only the fractions with dye trapped inside the liposomes were used for the assay. Liposomes with good dye incorporation were added to a 25 mM Hepes pH 8, 150 mM NaCl, 2 mM CaClbona fide ion channel activity using both planar lipid bilayer and liposome patch clamp techniques.Our previous studies mapped NSP4 viroporin activity to a 44-amino acid peptide corresponding to residues 47–90, and this region contains a cluster of basic residues and a predicted amphipathic α-helix, which are signature viroporin motifsWe tested the wild-type NSP4 VPD (VPD-WT) for ion channel activity using two well-established electrophysiology techniques, planar lipid bilayer and patch-clamp. In both techniques, we captured two unique electrophysiological signatures that characterize VPD-WT . Figure On occasion, the two types of kinetic behaviors could be seen within the same recording, as shown in bona fide ion channels. In conditions of 150 mM KCl, the unitary conductance of VPD-WT ranged from 40 to 65 pS.Altogether, we obtained 109 planar lipid bilayer recordings and 54 patch clamp recordings that showed that the VPD-WT peptide alone is capable of conducting monovalent ions. In ~70% of the recordings, VPD-WT displayed a discrete unitary level of conductance, as seen in canonical + (EK) and +58 mV for Cl− (ECl). Thus, the I/V plot of an ideal K+-selective channel would intersect the abscissa (0 pA) at −58 mV, while that of an ideal Cl−-selective channel would intersect the abscissa at + 58 mV. The intersect of the I/V plot with the abscissa is called the “reversal potential”. For NSP4 VPD, the 1:10 KCl gradient shifted the reversal potential to −39 mV, closer to EK and away from ECl. This demonstrated that NSP4-VPD selectively conducted cations (K+) over anions (Cl−). With this data, we used the Goldman-Hodgkin-Katz equationPreviously studied viroporins are generally cation selective with little conductivity of anions2+, we predicted that NSP4 should be able to conduct Ca2+ ions. To test this, we recorded the activity of VPD-WT in patch-clamp in symmetric buffer A conditions [buffer A  in both the bath and the pipette], and then perfused buffer CA (containing 75 mM CaCl2) into the bath. If the channel is able to conduct Ca2+, in these asymmetric buffer conditions opening transitions should be observed when a negative pipette voltage is applied to draw Ca2+ into the pipette. 2+, the sole cation able to sustain a current at negative pipette potentials  showed that the reversal potential shifted only slightly (~7 mV) in asymmetric conditions. Therefore, the channel does not appear to be more selective for K+ than Ca2+.Given that VPD-WT is cation-selective and that a strong correlation has been established between NSP4 and elevation of cytoplasmic Catentials . The contentials , and the2+ ions152+. Therefore, we tested whether VPD-WT was also capable of sustaining Ba2+ currents, by introducing buffer BA into our patch-clamp configuration in symmetric conditions. 2+ conditions, indicating that the channel can also conduct Ba2+ ions. The use of Ba2+ did not result in more patches with the well-defined openings, and fast current fluctuations were still ready observed (data not shown). We were able to determine a conductance of ~60 pS in symmetric 75 mM barium solutions from the well-defined openings observed in the traces. This value is similar to the conductance in 150 mM potassium solution , but in 73% (46% “mixed” + 27% “well-defined”) of the active patches with the cloud method. Therefore, the cloud method might be a slightly superior method for reconstituting NSP4 VPD peptide over the dehydration/rehydration method.In order to determine if the reconstitution technique affected the predominant kinetic state, we categorized our patches into 3 groups, according to the kinetic activity displayed throughout the experiment: “well-defined” openings only, “fast kinetics” only, and “mixed” (meaning the presence of both behaviors in the same experiment). E. coli lysis assay to confirm that the VPD-Mut lacks viroporin activity ] or soluble [NSP4 (95–146)]. The cells were separated into soluble, peripheral, and integral membrane fractions and analyzed by immunoblotting  did not cause an increase in fluorescence over time  in the presence of symmetric 150 mM Na+, suggesting that NSP4 VPD-WT peptide can sustain a significant Ca2+ flux even in the presence of high monovalent cation levels, as would occur between the ER lumen and cytoplasm of RV-infected cells.Finally, we examined the ion channel activity of the VPD-WT and VPD-Mut peptides. Unfortunately, due to low reconstitution efficacy, we were not able to reconstitute the VPD mutant (VPD-Mut) in liposomes suitable for patch clamp. Since NSP4 is localized to the ER membrane, the ability to conduct Ca2+ is induced by two different forms of NSP4 that produce distinct Ca2+ signals. Extracellular NSP4 (eNSP4) is a secreted enterotoxin that consists of a proteolytic cleavage product of the NSP4 cytoplasmic tail and elicits a phospholipase C (PLC)-dependent Ca2+ flux through interaction with cell surface receptors2+ through its viroporin activity2+ released upon stimulation of cells with known Ca2+ agonists , indicating that NSP4 increases the basal leakiness of the ER, possibly by ion channel activity132+-conducting ion channel.During a RV infection, elevated cytosolic Cabona fide ion channel, since it displays discrete levels of conductance. Interestingly, the NSP4 VPD peptide was soluble in water, which is distinct for other reported viroporins , and we speculate that the aqueous solubility of this peptide may have contributed to the relatively low reconstitution efficiency observed. The NSP4 VPD channel was capable of conducting K+, Ca2+, and Ba2+ ions and shows a strong selectivity for cations over anions, but interestingly, it was not selective for Ca2+ over K+ ions. Thus, like most viroporins, NSP4 is a non-selective cation channel with stochastic gating events; however, the permeability of VPD-WT for divalent ions sets it apart from many other viroporins, which are generally monovalent cation selective with low divalent cation permeability342+ selectivity is likely not an impediment to the release of ER Ca2+ in RV-infected cells because the 1000–5000 fold Ca2+ gradient across the ER membrane enables a sustained Ca2+ current even through a non-selective channel. The ability of NSP4 VPD-WT to conduct Ca2+ into dye-loaded liposomes in the presence of 150 mM Na+ supports this conclusion. These data fit with our previous observation that the ER Ca2+ sensor STIM1 and the store-operated calcium entry (SOCE) pathway are activated in RV-infected and NSP4-WT expressing cells, but not in cells expressing the viroporin-defective NSP4 mutant2+ in the liposome assay. Thus, our data indicates that the ion channel activity of the NSP4 VPD is mechanistically responsible for the PLC-independent elevation of cytosolic Ca2+ by both releasing ER Ca2+ and through this depletion of ER Ca2+, inducing the activation of SOCE through the plasma membrane.The electrophysiology studies reported here show that the NSP4 VPD is indeed a 2+ when expressed in cells and therefore fails to induce autophagosome formation62+ into liposomes, which could have been caused by either a defect in VPD-Mut membrane insertion or the formation of an inactive channel complex in the membrane. However, our previous study identified the NSP4 pentalysine domain, not the amphipathic domain, as the critical motif for transmembrane insertion of NSP42+, our data indicates that the mutant forms a defective Ca2+ channel complex in the membrane.We previously demonstrated that the NSP4 viroporin-defective mutant is unable to elevate cytosolic Ca2+ ions, takes on multiple oligomeric forms, and associates with several cellular and viral proteins232+ binding site (aa120/123), which was recently shown to depend on the oligomerization state of the CCD and is regulated by changes in pH232+ release channels are regulated by cytosolic Ca2+, but thus far, no Ca2+-dependent regulatory domain has been identified in a viroporin. In contrast, there is precedent for pH-mediated regulation of viroporins, since gating of the M2 viroporin from influenza A virus and the p7 viroporin from some hepatitis C virus strains are regulated by the pH of the endocytic compartment252+ levels during the course of a RV infection.This study also showed that the kinetics of the VPD-WT is predominantly fast, with frequent transient openings. This kinetic behavior is similar to that reported for other viroporins such as HCV p72+ by NSP4 ion channel activity is critical for productive RV replication through activation of a Ca2+-dependent autophagy pathway2+ would block RV replication. The well-defined opening events of NSP4 display a conductance in the range of ~40–60 pS similar to the conductance of L-type calcium channels, and it is possible that NSP4 could be blocked by L-type calcium channel blockers such as the benzothiazepine, dihydropyridine, or phenylalkylamine classes of blockers, which are widely used clinically, even in children2+ influx into RV-infected cells, which was predicted to be through blocking plasma membrane-localized host Ca2+ channels; however, it is possible these drugs may also inhibit NSP4 functions2+ is critical for Cl− secretion both from RV-infected enterocytes and through activation of the enteric nervous system via increased serotonin secretion29The elevation in cytosolic Ca2+ levels are RV NSP4 and enterovirus 2B, and thus far their effects on Ca2+ levels have been characterized using fluorescent Ca2+ imaging techniquesbona fide ion channel activity and can conduct Ca2+. Recent studies show that other viroporins, previously characterized as being monovalent cation selective, can also conduct Ca2+ under some conditions. The E protein of severe acute respiratory syndrome (SARS) coronavirus can conduct Ca2+ when reconstituted into membranes that mimicked those of the ERGIC/Golgi compartments2+ conductivity under the right experimental conditions, and exploitation of host Ca2+ signaling pathways by viroporin activity may be more common than currently appreciated.The best characterized viroporins that affect cellular CaHow to cite this article: Pham, T. et al. The Rotavirus NSP4 Viroporin Domain is a Calcium-Conducting Ion Channel. Sci. Rep.7, 43487; doi: 10.1038/srep43487 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H+ for cytoplasmic Ca2+, respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H+/Ca2+ exchangers, Gdt1 also promoted growth in high Ca2+ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together with Gdt1.Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H The glyproducts . Nucleot2+ or Mn2+ ions for maximal activity  to promote Ca2+ influx, elevation of cytosolic free Ca2+ concentrations, and replenishment of the secretory Ca2+ pools  through 0 (wild-type growth) to −5 (greatly decreased or no growth). The screen was repeated twice independently. Of the 107 mutant strains that consistently exhibited a significant response to at least one condition, 84 were found to exhibit elevated Ca2+ influx in YPD medium or YPD medium plus FK506  confirmations.The yeast knockout collection in strain BY4741 background  were streaked for single colonies on agar YPD medium, and 36 single colonies were picked, grown further on the same medium to allow spontaneous mutations to accumulate, and then incubated on noble agar YPD medium containing 5 mM succinic acid and 111 mM CaCl2 for several days. A single Ca2+-tolerant colony was picked in each case, purified by restreaking, and then mated with one another in all possible combinations. The resulting diploids were reanalyzed for Ca2+ tolerance. This complementation test indicated that 26 haploid strains contained dominant mutations in unknown genes and 10 haploid strains contained recessive mutations all in the same gene. The 10 recessive mutants were fortuitously found to exhibit hypersensitivity to tunicamycin. One recessive mutant was transformed with a low-copy plasmid library containing fragments of yeast genomic DNA, and nine transformants were replica plated to YPD plus 2 µg/ml tunicamycin to select for strains bearing complementing plasmids. Two different plasmids overlapping the ERD1 gene and several nearby genes were recovered and found to reverse both the tunicamycin hypersensitivity and the Ca2+ tolerance phenotypes upon retransformation into the recessive mutant strains. ERD1 was identified as the defective gene in the recessive mutants by (1) introducing an erd1∆ null mutation into the K1357 strain and obtaining both the tunicamycin hypersensitivity and the Ca2+ resistance phenotypes and (2) demonstrating non-complementation between the erd1∆ null strain and the spontaneous recessive mutant strains. Similar erd1∆ knockout mutations were also introduced by transformation into several other strains; all strains utilized in figures are listed in Supplemental Material, Table S2. Table S1 lists additional strains in the W303 background, their genotypes, and quantitative analyses of their functions in Ca2+ tolerance.To identify spontaneous mutants that increase Ca2 in 96-well dishes with and without 1 µg/ml FK506, mixed, and incubated at 30° for 24 hr without shaking. The cells were resuspended by vortex mixing and optical density was measured at 650 nm using a microplate spectrophotometer (Molecular Devices). The concentration of Ca2+ causing a 50% decrease in maximal optical density (the IC50) and the concentration of Pi permitting growth to 50% maximal optical density (the ED50) were calculated for each strain by non-linear regression using the sigmoid equation with four parameters . The averages from two independently generated strains of the same genotype (±SD) were calculated and plotted. In Table S1, the coefficient of variation (CV) is listed instead of SD, and the data from two separate experiments (A and B) were normalized to a third reference dataset using linear regression after log-transformation.Yeast strains were grown to saturation at 30°, typically overnight, in YPD medium for ion tolerance assays or in synthetic complete (SC) medium for Pi dependence assays. They were then diluted 1:1000 into fresh media containing 5 mM succinic acid and various concentrations of CaClPMC1-lacZ reporter gene carried on the high-copy plasmid pKC190  and chloroform, and assayed for β-galactosidase activity using o-nitrophenyl-β-galactoside as substrate as described previously (The d pKC190  was traneviously .Yeast strains were grown overnight to saturation in SC medium (7.35 mM Pi) and then diluted 1000-fold into fresh SC medium containing tracer quantities (∼30 µCi per ml) of fresh [32]Pi (Perkin Elmer). After 12 hr of incubation with shaking, the log-phase cells were pelleted and washed five times with 5 ml of ice-cold Pi-free SC media to remove all unincorporated [32]Pi from the medium. The cells were then resuspended in Pi-free SC media and incubated on ice or at 30° and sampled at various times. After centrifugation at 15,000 rpm for 1 min, the cell-free supernatant was analyzed by liquid scintillation counting and by thin-layer chromatography (TLC), by spotting 3 μl of sample supernatant onto polyethylenimine (PEI)-cellulose TLC plates, which was then developed in 1 M LiCl with 10 mM HEPES, pH 7.5. The TLC plate was imaged by autoradiography.Cells were lysed via fast alkaline lysis in 0.5 M NaOH with 1.85% beta-mercaptoethanol (BME) in ice water for 10 min. The protein was then isolated through precipitation by addition of trichloroacetic acid (TCA) to a final concentration of 10% (w/w). Equal volumes of protein isolate and 2× SDS sample buffer  were then combined, and the samples were incubated at 37° for 30 min. Proteins were processed by separation on a 10% SDS/polyacrylamide gel electrophoresis (PAGE) gel and western blotting, as described previously . Blots wt-tests were implemented on many datasets, as indicated in the figures .Student’s All yeast strains and plasmids are archived and available upon request. Raw data files used to generate figures and tables are also archived and available upon request. All requests should be made to the corresponding author.2+ environments depends mostly on the vacuolar Ca2+ ATPase (Pmc1) when calcineurin is functioning, and mostly on the vacuolar H+/Ca2+ exchanger (Vcx1) when calcineurin has been inhibited or mutated . A similar genetic screen using cyclosporine instead of FK506 to inhibit calcineurin yielded partially overlapping results (2), and then filtering out the candidates that had exhibited elevated Ca2+ uptake in response to FK506 in a previous study  exhibited hypersensitivity to Ca2+ but not Ca2+ plus FK506, but were not studied further.The ability of yeast cells to proliferate in high Ca mutated . To sear1∆-like” . As expe of Pmc1 . Two othvcx1  may be deficient in positive regulators of Vcx1 or in novel Ca2+ transporters. Rav2 forms a complex with Rav1 and promotes assembly of V1 and V0 sectors of the vacuolar form of the V-ATPase  while preserving the Golgi/endosomal forms exhibited no hypersensitivity to high Mg2+ and strong hypersensitivity to high Ca2+ and high Ca2+ plus FK506 conditions  were compared for all eight strains. Relative to the wild-type parent, the pmc1∆ vcx1∆ vph1∆ triple mutant was ∼19-fold more sensitive to Ca2+ in the medium (2+ strongly (4.2-fold), whereas restoring Vcx1 had little effect (1.1-fold) in the presence or absence of Pmc1. Restoring Vph1 function had no significant effect in the cells that lack both Pmc1 and Vcx1 (1.05-fold), but had strong effects on cells expressing only Vcx1 (2.2-fold) or Pmc1 (4.4-fold). Therefore, Pmc1 activity was partially dependent on a functional V-ATPase in the vacuolar membrane, and the relatively low Vcx1 activity detectable in pmc1∆ mutant backgrounds was totally dependent on the vacuolar V-ATPase.To investigate how the vacuolar V-ATPase affects Pmc1 and Vcx1 function, a pmc1∆ vcx1∆ vph1∆ triple mutant became 2.7-fold more sensitive to Ca2+ in the presence of FK506 than in the absence of FK506, presumably owing to diminished function of the Crz1 transcription factor and therefore diminished expression of the Pmr1 Ca2+/Mn2+ ATPase in the Golgi complex when calcineurin is inhibited. Restoring Vcx1 function alone had little effect (1.3-fold increase) in the presence of FK506, whereas restoring Pmc1 function alone had strong effects (5.2-fold increase) despite a lower expression of Pmc1 in the absence of calcineurin  in the absence of both calcineurin and only when Pmc1 was functioning. All these findings were reproducible in a replicate experiments and show, for the first time, that Vcx1 can antagonize the function of Pmc1 when the vacuoles lack acidification by the V-ATPase and when the cytoplasm lacks calcineurin signaling. Such antagonism was expected, because H+/Ca2+ exchangers are generally thought to operate in “reverse mode” in conditions where the luminal H+ concentration is low and the Ca2+ concentration is high, potentially causing futile cycles between Vcx1 and Pmc1 in the absence of Vph1. Reverse-mode operation of Na+/Ca2+ exchangers is well established (vph1∆ mutants with FK506) did not occur in the absence of FK506 suggests that calcineurin regulates both forward and reverse modes of Vcx1 through a process that is independent of the V-ATPase.To test whether the vacuolar V-ATPase mediates the interaction between calcineurin and Vcx1, the same eight mutant strains were reanalyzed in the presence of FK506  but strongly diminished Ca2+ tolerance in WT, pmc1∆, vcx1∆, and pmc1∆ vcx1∆ backgrounds when FK506 was present . Thus, in the absence of calcineurin signaling, Gdt1 strongly promoted Ca2+ tolerance independent of both Vcx1 and Pmc1, and likely represents a new class of Ca2+ transporter that is partially inhibited by calcineurin.The nditions  and ther2+ tolerance experiments described above in crz1∆ cmk2∆ double mutant and single mutant backgrounds. This experiment ruled out Crz1 and Cmk2 as possible intermediaries in the inhibition of Gdt1 by calcineurin (see Table S1). To test whether calcineurin signaling could diminish expression of Gdt1 or alter its mobility on SDS-PAGE, western blots were performed on cells containing a tandem affinity purification (TAP) epitope tag, integrated at the 3′ end of the chromosomal GDT1 gene . After exposure of the cells to 200 mM CaCl2 with or without 1 µg/ml FK506 for 12 hr, the Gdt1-TAP band maintained the same intensity and migration on the gel , suggesting that Gdt1 cannot remove toxic Mn2+ from the cytoplasm. The gdt1∆ mutant exhibited a strong hypersensitivity to BAPTA relative to the wild-type parent strain, and the gdt1∆ pmr1∆ double mutant exhibited extreme hypersensitivity to BAPTA that was significantly greater than the pmr1∆ mutant . This lethality of FK506 suggests that calcineurin-dependent upregulation of Pmc1 and relocalization to the Golgi complex . In this vph1∆ vcx1∆ double mutant background, the loss of Gdt1 caused a more than 10-fold decrease in BAPTA tolerance and the further loss of Stv1 had little additional effect . Of 36 independent spontaneous mutations, 26 were found to be dominant in heterozygous diploids. The GDT1 coding sequence from all 26 strains was amplified by PCR and sequenced. No mutations were identified in the GDT1 coding sequences. The mechanism of Gdt1 regulation by calcineurin remains unknown and may involve several unknown intermediary steps (see Discussion).If activated calcineurin inhibits Gdt1 function, it may be possible to isolate variants of Gdt1 that are hyperactive when calcineurin is fully functional, similar to the hyperactive Vcx1-D mutants that have been isolated previously . To focu2+-resistant mutants were found to be hypersensitive to tunicamycin, an inhibitor of N-glycosylation reactions in the ER , and that this effect was abolished if Gdt1 were eliminated (Table S1). Erd1 actually increased Ca2+ tolerance or was neutral in most other conditions. These results are consistent with models where Erd1 selectively diminishes the forward function of Gdt1 in the Golgi complex.In addition to the 26 dominant mutants, 10 independent recessive mutants were recovered, and these mutants defined a single complementation group. All of the recessive Can the ER , and we erd1∆ mutants fail to recycle Pi to the cytoplasm, and the backlog of Pi in the lumen increases the buffer capacity for Ca2+ while also interfering with normal glycosylation and sorting reactions. To test the hypothesis that Erd1 recycles the Pi byproduct of glycosylation from the lumen of secretory organelles, several experiments were performed.Erd1 contains an EXS domain similar to that of human XPR1 and plant PHO1 proteins, which recently have been shown to export Pi from the cell , it is pi.e., the ED50) was about 1.5-fold higher for erd1∆ mutants compared with the wild-type control strain  supplemented with tracer amounts of [32]Pi, chilled, and the cells were washed extensively in fresh medium lacking Pi. The temperature was then raised to 30° and aliquots were removed at different times, centrifuged to pellet the cells, and the cell-free supernatants were collected and analyzed by liquid scintillation counting and by TLC. In the first 0.25 hr of incubation at 30°, both erd1∆ and wild-type strains exported similar amounts of radioactivity. However, the erd1∆ mutant exported strikingly more Pi than wild type over the next 1.75 hr of incubation  or the luminal nucleoside triphosphate diphosphatases (Gda1 and Ynd1) are lethal , and in a unicellular relative of metazoans (the choanoflagellate Monosiga brevicollis), but not in metazoans. This suggests that the Erd1 subfamily of ESX-domain proteins originated prior to the divergence of fungi and animals but were uniquely lost from the metazoan lineage. This loss in metazoans could have been enabled by the gain of another Pi transporter in the Golgi complex. Alternatively, the benefits of Pi recycling from the Golgi complex may have diminished in metazoans, where the loss of Pi to the extracellular fluids is not harmful or is potentially even beneficial for Pi storage, for pH buffering, or for creating extracellular structures.The evolutionary origins of Erd1 also may be instructive about its functions in cell physiology. Our searches of protein databanks and multiple sequence alignments (not shown) reveal orthologs of Erd1 in virtually all species of fungi, in a unicellular relative of fungi (the nucleariid erd1∆ mutants of yeast exhibit striking deficiencies in secretory protein glycosylation and in sorting by the H/KDEL receptor (Erd2), which ordinarily retrieves many ER-resident molecular chaperones and enzymes that have escaped the ER  also co- or countertransports some other molecule together with Pi. Direct transport studies using liposomes with purified and reconstituted Erd1 protein and other biochemical experiments will be needed to test these possibilities.In addition to defects in Pi recycling, 2+ environments independent of the known Ca2+ transporters  and only when the Golgi complex receives sufficient acidification from the V-ATPase. In the absence of all V-ATPase activity, Gdt1 seemed to partially undo the work of the other transporters and to have the opposite effects on cell growth and on calcineurin activation in the cytoplasm. By showing that Gdt1 can operate in reverse mode when the V-ATPase is eliminated, we add strong experimental support to the hypothesis that Gdt1 catalyzes H+/Ca2+ exchange in the Golgi complex , suggesting that Gdt1 may not transport significant levels of Mn2+. Consistent with these findings, the partial rescue of glycosylation defects in pmr1∆ mutants by supplemental Ca2+ depended on Gdt1, but the partial rescue by supplemental Mn2+ occurred independent of Gdt1  result in synthetic lethality in the human HAP1 cell line  by which calcineurin regulates the function of Gdt1 and Vcx1 remain unknown, as neither protein undergoes changes in expression or mobility on SDS-PAGE upon activation/inhibition of calcineurin. Our finding that calcineurin still inhibited reverse-mode activity of Vcx1 in the absence of Vph1 suggests that neither the vacuolar V-ATPase nor luminal H+ are key mediators of this regulation. We also ruled out Erd1 as an intermediary of Gdt1 inhibition by calcineurin, as this regulation persisted in erd1∆ vcx1∆ strains (Table S1). Because Gdt1 and Vcx1 both seem to promote H+/Ca2+ exchange, it is tempting to speculate that calcineurin regulates the pH of the cytoplasm or organelles in high Ca2+ conditions, which would alter the ability of transporters to bind Ca2+. Indeed, new evidence suggests that calcineurin may downregulate the plasma membrane H+ pump Pma1 , potentially causing acidification of the cytoplasm and diminishing forward Ca2+ transport by H+/Ca2+ exchangers.We also provide evidence that activated calcineurin can inhibit the forward-mode activity of Gdt1 2+ from the cytoplasm, both proteins have the potential to form double-negative feedback loops with calcineurin , as observed previously during the switching between high- and low-affinity Pi transporters in yeast . It will be interesting to determine precisely how and why calcineurin inhibits Gdt1 and Vcx1 functions in vivo, and whether such regulation contributes to the unexplained “bursts” of free Ca2+ elevation and calcineurin signaling that have been observed through real-time imaging in single yeast cells  and casein micelles (rich in Ca2+ and Pi) in the Golgi complex during lactation (+ byproduct in exchange for Ca2+ may be exceptionally important. Interestingly, to potentially meet this demand, expression of TMEM165 mRNA and protein becomes massively increased in alveolar epithelial cells just as milk production begins (A better understanding of Hactation , retrievwww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300339/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.


We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.Inositol 1,4,5-trisphosphate (IP  •3-evoked Ca2+ releaseCyclic AMP directly potentiates IP•2+ stores released by IP3 alone or IP3 with cAMP are functionally independentThe Ca•3 receptors in a discrete ER Ca2+ storeCyclic AMP unmasks high-affinity IP•2+ stores increases signaling versatilityIndependent regulation of discrete Ca 3-evoked Ca2+ release. Konieczny et al. find that IP3 alone and IP3 with cAMP release Ca2+ from independent stores within the endoplasmic reticulum. Compartmentalized Ca2+ stores increase the versatility of IP3-mediated Ca2+ signaling.Cyclic AMP directly potentiates IP GPCRs evoke cAMP formation by stimulating adenylyl cyclases (ACs), whereas most GPCR-evoked Ca2+ signals result from stimulation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3). IP3 then evokes Ca2+ release from the endoplasmic reticulum (ER) through IP3 receptors (IP3Rs)  comprise the largest class of cell-surface receptors, and they endow cells with the ability to respond to diverse extracellular stimuli. However, most signaling from GPCRs proceeds through a very small number of intracellular messengers, among which 3′,5′-cyclic AMP (cAMP) and Ca (IP3Rs) A (Fosket (IP3Rs) . At leastogether . Finallytogether .3Rs can be phosphorylated by cAMP-dependent protein kinase (PKA) and, at least for IP3R1 and IP3R2, this increases their IP3 sensitivity   A (Kurian (EPACs) . This po cytosol . We haves to IP3 A , carbachol (CCh) evoked a concentration-dependent increase in [Ca2+]i (intracellular free Ca2+ concentration)  in HEK cells stably expressing type 1 human parathyroid hormone (PTH) receptor (HEK-PR1 cells) (3 muscarinic acetylcholine receptors (M3R) of HEK293 cells stimulate Ca2+ release from intracellular stores through IP3Rs  B and 1C.gh IP3Rs . Neithers of CCh B–1G. The93 cells   or submaximal (30 nM) concentration of PTH. The results demonstrate that for CCh concentrations that evoked Ca2+ signals of very different amplitudes (∼20–340 nM), the additional increase in [Ca2+]i evoked by PTH was almost invariant, but larger for the maximal concentration of PTH (Δ[Ca2+]i ∼240 nM) than for the submaximal PTH concentration (Δ[Ca2+]i ∼170 nM)  D. Similaal level H and 1I.3Rs: the maximal increase in [Ca2+]i evoked by Mfm was only 36% ± 1% of that evoked by CCh  is a partial agonist of Md by CCh A. Nevertwith CCh A and 2B.with CCh C and 2D.me cells E. Nevert and ATP E.3Rs to IP3, its effects should be most pronounced at the lowest CCh concentrations  across most CCh concentrations  is unexptrations F. The sa stimuli A–2E. It 2+]i, the additional Ca2+ release evoked by PTH  is similar. We can envisage two possible explanations for these observations. It may be that all stimuli release Ca2+ from a shared Ca2+ store, and the consistent responses to PTH then reflect a balance, as the CCh concentration increases, between the declining content of the Ca2+ store and a compensating increase in the sensitivity of a larger number of IP3Rs  decreased with time   in Ca± 1 min) A. After response B and 3C.2+ content of the CCh-sensitive stores. If, for example, IP3Rs were regulated by luminal Ca2+, then CCh-evoked Ca2+ release might terminate before the stores were empty. However, when cells were treated with 1 μM thapsigargin to inhibit the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and so unmask a Ca2+ leak from the ER, the rates of decline of the response to 1 mM CCh  and of the Ca2+ content of the stores assessed by addition of 1 μM ionomycin (t½ = 3.0 ± 0.3 min) were indistinguishable  similar to that evoked by addition of 1 mM CCh (110 ± 7 nM) . Analysis of single HEK-PR1 cells using the same protocol established that the very different effects of depleting CCh-sensitive Ca2+ stores on subsequent responses to CCh or PTH were not due to cellular heterogeneity (Addition of PTH (100 nM) to HEK-PR1 cells stimulated for 2 min with 30 μM CCh in Ca ± 7 nM) A–4C. How ± 7 nM) B and 4C.ogeneity E.3 . During sustained exposure to 30 μM CCh, the increase in [Ca2+]i evoked by subsequent addition of PTH was abolished when methylatropine was added with the PTH   , and it 2+ entry (SOCE) is required for resynthesis of the pool of phosphatidylinositol 4,5-bisphosphate that sustains IP3 production during activation of leukotriene receptors  or maximal (1 mM) concentration of CCh to deplete the CCh-sensitive Catrations A. These 2+ stores released by CCh alone or CCh with PTH  unmasks IP3R-binding protein released by IP3), is an endogenous IP3R antagonist (S-adenosylhomocysteine-hydrolase (AHCY), binds cAMP  and Orai at ER-plasma membrane junctions  with the amplitude of the subsequent sustained increase in [Ca2+]i (SOCE) revealed that the relationship between the two Ca2+ signals was indistinguishable for cells stimulated with the different stimuli   . Both Can SERCAs . Becausen SERCAs , allowinn SERCAs , we conssigargin .2+ sensor   shows that neither organelle responded selectively to PTH   targetedpparatus A to measthen PTH B. CCh anTH  or CCh with PTH (t½ = 3.9 ± 0.2 min)  B, sugges2+ store , these r another C, and thd stores D.2+-free HBS were incubated for 60 min with 30 μM CCh to empty the CCh-sensitive Ca2+ stores; the cells were then rapidly washed in Ca2+-free HBS to remove CCh, and the responses to CCh alone or CCh with PTH in Ca2+-free HBS were assessed during this recovery period. At the end of the 60 min incubation with 30 μM CCh, the response to addition of 1 mM CCh had declined to 1% ± 1% (n = 3) of the initial response, consistent with previous results . However, the response to addition of PTH after CCh remained constant over the entire recovery period , but these IP3Rs are unmasked only in the presence of cAMP. We have not established the identities of the independent Ca2+ stores, although it is clear that IP3R2, which we showed to be important for responses to PTH  in single cells and populations of fluo-4-loaded HEK-PR1 cells were performed as previously described (HEK-PR1 cells  were culescribed . Intraceescribed  (Supplemescribed .D = 24 μM), red genetically encoded Ca2+ sensor (LAR-GECO1) was used to record the luminal [Ca2+] within the ER ([Ca2+]ER using ER-LAR-GECO1) (2+]GA using Golgi-LAR-GECO1). Details are given in the A low-affinity (KR-GECO1)  or withiBacMam viruses were used to express IRBIT and IRBIT-S68A in HEK-PR1 cells. Cells were transfected with siRNAs to reduce IRBIT expression in HEK-PR1 cells .2+]i and sensitivities to CCh and PTH. Hence, all statistical comparisons use observations from matched analyses. For each experiment, the concentration-effect relationship was fitted to a logistic equation (GraphPad Prism version 5). From each experiment, pEC50  and the maximal response were obtained and used for statistical analyses. Most graphs show mean results from several experiments, but values  were computed from individual experiments before pooling for statistical comparisons.The experiments reported were completed over a prolonged period during which there was some variation between absolute values for changes in [Ca2+ indicators. C.W.T. supervised the project and contributed to data analysis. C.W.T with V.K and D.L.P. wrote the paper. All authors contributed to review of the paper.V.K. performed experiments. S.C.T. contributed to fluorescence experiments. S.M. performed western blot (WB) and analyses of STIM1. D.L.P. contributed to design and analysis of targeted Ca


With the cytosolic Ca2+ chelated by EGTA and BAPTA or extracellular Ca2+ replaced by Ba2+, effects of bufadienolides  and cinobufagin (CBG)) and cardenolides (ouabain (Oua) and pecilocerin A (PEA)) on the L-type calcium current  were recorded in heterologous expression Cav1.2-CHO cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). BF and CBG demonstrated a concentration-dependent (0.1 to 100 µM) ICa,L inhibition  without and with the NKA activity blocked by 10 µM Oua. BF significantly shortened the action potential duration at 1.0 µM and shortened the extracellular field potential duration at 0.01~1.0 µM. On the other hand, BF and CBG at 100 µM demonstrated a strong inhibition (≥40%) of the rapidly activating component of the delayed rectifier K+ current (IKr) in heterologous expression HEK293 cells and prolonged the APD of the heart of day-3 Zebrafish larva with disrupted rhythmic contractions. Moreover, hESC-CMs treated with BF (10 nM) for 24 hours showed moderate yet significant prolongation in APD90. In conclusion, our data indicate that CGs particularly bufadienolides possess cytosolic [Ca2+]i- and NKA inhibition- independent proarrhythmic potential through ICa,L and IKr inhibitions.The current study explored the Na Members of the cardiac glycoside family share a similar structural motif consisting of a basic perhydrophenanthrene nucleus and an unsaturated lactone ring at C-17 position. The lactone moiety defines two main classes of these compounds. Bufadienolides  and cinobufagin (CBG)) and possess a six membered butenolide ring whereas cardenolides (ouabain (Oua) and pecilocerin A (PEA)) possess a five membered pentadienolide ring2.Cardiac glycosides (CGs) are selective inhibitors of sodium-potassium adenosine triphosphatase  of the L-type Calcium channels (LTCC) and lead to the suppression of the LTCC currents 12, which could be responsible for the shortening of cardiac action potential (AP) duration (APD) in cardiac myocytes of various animal species16 and the shortening of the cardiac field potential (FP) duration of human induced pluripotent stem cell-derived cardiomyocytes17. Moreover, increased [Ca2+]i. and SR Ca2+ load by NKA inhibition could activate Calcium-Calmodulin Kinase II (CaMKII)-induce phosphorylation of the ryanodine receptor (RyR) on the sarcoplasmic reticulum (SR) of cardiac myocytes and enhanced SR Ca2+ leak and exacerbate spontaneous cardiac activity19. However, questions remain as whether NKA inhibition and [Ca2+]i accumulation by CGs is fully responsible for the pro-arrhythmic electrophysiological changes. The dissociation between inotropic and proarrhythmic actions of CGs indicates that the proarrhythmic effects of CGs may not result solely from NKA inhibition21. For example, Chan Su (toad venom) and its key ingredient BF have demonstrated more potent cardiotoxicity over Oua as additional proarrhythmic effects were achieved by Chan Su or BF after the Na+/K+-ATPase activity was fully blocked by Oua22.However, CGs have long been known for possessing strong proarrhythmic effects that could cause syncope and virtually all forms of cardiac arrhythmias in human associated with impaired atrial-ventricular conduction and enhanced automaticity2+]i- and NKA inhibition- independent proarrhythmic ionic mechanism of CGs. Bufadienolides (BF and CBG) and cardenolides (Oua and Pecilocerin A (PEA))  with different NKA inhibition potencies24, were assayed with the cytosolic Ca2+ chelated, extracellular Ca2+ or K+ depleted and NKA activity blocked. Effects of CGs on major cardiac ion channel currents and corresponding repolarization durations were assessed in heterologous expression systems, human embryonic stem cell (hESC)-derived cardiomyocytes (hESC-CMs) and zebrafish heart, respectively. Our data confirmed that, under physiological/toxicological concentrations, CGs particularly Bufadienolides like BF could either inhibit LTCC independently from intracellular [Ca2+]i and NKA-inhibition, or, inhibited hERG at both high and low (prolonged effect) concentrations.CG-induced cardiotoxicity remains to be a threat to human health and it may hamper their clinical potentials as hypertonic and anti-cancer drugs. Hence, it is important to fully understand the proarrhythmic effect and mechanisms of CGs. Here, we explored the potential i in neonatal rat cardiac myocytes with NKA activity fully inhibited by 10 µM Oua20. Similarly, we observed that on hESC-CMs pre-incubated with 10 µM Oua for 3 minutes, BF (10 µM) reduced the [Ca2+]i transients amplitude  and duration at 50% and 80% recovery (CaD50 and CaD80), and increased time to peak . At 1.0 µM, all CGs tended to shorten APDc  and it was more evidenced with BF and CBG. Yet only the effects of BF were statistically significant  and beating frequency were noted. BF (10 µM) treatment for 1–2 minutes achieved more remarkable changes in APs, followed by polymorphic arrhythmia-like AP waveform at 5 minutes and AP firing ceased at 5~8 minutes (data not shown).To correlate the CG-induced changes in cardiac ion currents with transmembrane activities, APs were recorded in single hESC-CMs under cytosolic i was able to reduce APD  alone failed to positively shift MDP and shorten APD  alone  on APs confirmed the experimental findings , an potential equivalent of ICa,L  model (2011) of non-diseased human midmyocardial ventricular myocytesAPD Fig. . Yet redAPD Fig. . Next, rone Fig.  or with NaK Fig.  led to mngs Fig. . Interesa,L Fig. .Figure 82+ largely chelated or extracellular Ca2+ depleted, bufadienolides demonstrated a cytosolic [Ca2+]i-independent ICa,L inhibition, accompanied by APD shortenings. With the NKA activities completely inhibited by Oua, effects of bufadienolides on ICa,L persisted. Further, a strong Ikr blockage in hERG-HEK cells and a prolonged APD in zebrafish larva were observed by BF and CBG at 100 µM, whereas prolonged APD was also observed in hESC-CMs treated with 10 nM BF for 24 hours. Lastly, results from computer simulation largely validated the experimental findings on the relationship between ion currents and APs.The current study identified the NKA inhibition-independent proarrhythmic mechanisms of CGs which involve key cardiac ion channel currents and transmembrane potentials. With the cytosolic CaICa,L inhibition could contribute to the greater proarrhythmic effects of BF and CBG compared with Oua and PEA. While it is known that 10 μM of Oua could totally blocked the NKA activities22 and BF and CBG are 4~7 folds more potent NKA blockers than Oua23, the more potent proarrhythmic effects of BF in APD shortening13 and [Ca2+]i transient suppression over Oua suggesting that bufadienolides could have NKA-independent proarrhythmic effects22. In the current study, we firstly observed cytosolic [Ca2+]i- or CDI- independent effects of BF and CBG on ICa,L inhibition and APD shortening. ICa,L was measured in the presence of 10 ~20 mM of EGTA in the pipette/internal solution and the APs were measured with 10 mM EGTA in the pipette solution, while ≥5 mM EGTA is sufficient to achieve a complete chelation of cytosolic free Ca2+ with [Ca2+]i up to 2.5 mM [Calculated by a Ca-EGTA Calculator v1.3 online from Maxchelator program of Stanford]. Hence EGTA could eliminate CDI due to CG-induced NKA inhibition29. In addition, BAPTA, the other calcium chelator we added to the intracellular solution (5 mM), could, by itself, fully block CDI29. Furthermore, our data showed that the ICa,L inhibitory effect of BF retained in Cav1.2-CHO cells with extracellular Ca2+ replaced by Ba2+, while depletion of extracellular Ca2+ is known to fully blocked CDI29. Secondly, we gathered evident to support that BF and CBG possess NKA inhibition-independent effects. The concentration-dependent ICa,L inhibition by BF and CBG peaked at 100 µM and it further suggests that BF and CBG are capable of inducing NKA inhibition-independent proarrhythmic effects as <2.5 µM of BF or CBG could be sufficient to fully block the NKA activities24. Next, we noticed that the effects of BF and CBG on ICa,L and [Ca2+]i transient persisted in cells incubated with 10 µM Oua. The rapid inhibition of ICa,L and shortening of APs by BF and CBG support direct effects on LTCC since the effects of NKA inhibition and CDI on APs could take longer time13. While CDI of ICa,L is known to accelerate the decay of currents without supressing the peak currents, our finding that BF, without/with NKA activity blocked by Oua, markedly inhibit peak ICa,L with magnitudes greater than that for the decay current  which could be behind the APD prolongation in zebrafish lava. However, the impact of a potential ICa,L inhibition, in completion with IKr inhibition, on APD in Zebra fish remains unclear. Result from computer simulation may suggest that APD is more sensitive to IKr inhibition. It appears that zebrafish is less sensitive to CG-induced cardiotoxicity probably due to inter-species variations and the arrhythmogenic concentration of CGs is less physiologically relevant. With more clinical relevant concentrations (the recommended digoxin concentrations for the treatment of chronic heart failure are around 0.8 ng/ml (1.2 nM) with a half-life of 36 hours and the toxic level is more than 2 ng/ml30), we demonstrated that CGs could prolongation APD and such an effect could be attributed to the chronic effects of low concentration CGs on hERG inhibition25.14 appeared to be less obvious in hESC-CMs  added in the pipette solution that could suppress CDI and minimize the APD shortening effect of CGs in hESC-CMs.The well-documented effect of CGs on depolarizing the MDP and shortening APD in isolated cardiac myocytesCMs Fig. . This diICa,L reduction. Such a discrepancy may reflect the limitations with the ORd model, which has been shown failing to simulate considerable changes in APs with marked inhibitions of INaK and ICa, L32. Compared with the moderate negative correlation between [Na+]i and APDs simulated by the ORd model, Grandi et al.33, using a mathematical model for Ca2+ handling and ionic currents in the human ventricular myocyte, demonstrated that Na+ loading could be a major determinant of ventricular APD shortening, and therefore that model could be a better choice for modeling effects of NKA block and ICa,L block.Effects of CGs on APD are supported by the simulated results in principle although only moderate APD shortening was simulated by the ORd human ventricular myocyte model (2011) in responds to a marked ICa,L and cardiac arrhythmias is supported by the clinical findings. For example, loss-of-function mutations in the LTCC have been associated with QT shortening and severe arrhythmia in patients with the Brugada syndrome34.While the arrhythmogenic potential associated with LTCC inhibition could be less understood compared with hERG inhibition, the association between reduced 2+]i and accelerate the proarrhythmic effects in the meantime.Overall, our data may explain the dissociation between the positive inotropic effect and the proarrhythmic effects of CGs. For example, the additional LTCC inhibition by BF over Oua may undermine the hypertonic effect of BF by attenuating the −1), where G is the conductance at various test potentials and was calculated from the peak current according to G = I/(V − Vrev), Vrev is the reversal potential obtained by extrapolating the linear part of the I/V curve to its intersection with the voltage axis. Gmax is maximum conductance; V1/2 and κ are half-activation voltage and the slope factor. The concentration-response data were fitted with Hill equation: I/Imax = 1/[1+ (D/IC50)n], where I is the peak current in various concentrations of compound, Imax is the maximal peak current, D is the compound concentration, IC50 is the drug concentration for 50% inhibition, and n is the Hill coefficient.Calcium channel current densities were obtained by dividing current amplitudes by membrane capacitances. Steady state inactivation variables of ICa,L was studied using a two-pulse protocol: a 250-ms pre-pulse (P1) at 0 mV from the holding potential of −50 mV followed by a variable recovery period and a 250-ms test pulse (P2) at 0 mV to assess the amount of current recovered. Each two-pulse sequence was separated by a 30 s interval. The time course of recovery for ICa,L was determined by fitting the data points to a single exponential function: I/Imax = 1 − exp(−t/τ), where Imax and I were the peak current at pre-pulse (P1) and test pulse (P2), respectively; t was the variable recovery time; τ was the recovery time constant.The time course of recovery from inactivation of 2 1.8, MgCl2 1, glucose 10, HEPES 10, adjusted to pH 7.4 with NaOH. Pipette solution contained (in mM): KCl 130, NaCl 5, MgCl2 1, MgATP 3, EGTA 10, and HEPES 10, adjusted to pH 7.2 with KOH. The parameters of APs include APD at 30%, 50% and 90% of repolarization , APA, MDP, and beating frequency were analyzed38. Cells were maintained at 35 °C by a temperature controller  during the recording of APs. The APDs were corrected by the beating frequency (APDc) with Fridericia’s formula 39.APs were recorded under current-clamp mode in normal Tyrode’s solution contained (in mM): NaCl 140, KCl 5.4, CaCl2+]i transients were recorded in hESC-CMs using a LSM-710 laser scanning confocal microscope  with a 40×, 1.3 numerical aperture oil immersion objective and axial resolutions of 1.5 µm38. Briefly, hESC-CMs were loaded with 2 μM Fluo-8 AM  for 15 min at 37 °C, and recorded in normal Tyrode’s solution. Fluo-8 was excited at 488 nm, and fluorescence emission was measured at 505 nm. Images were acquired in the line-scan (X-T) mode with 512 pixels  per line at a rate of 3 ms per scan. The [Ca2+]i transients were analyzed using a modified version of MATLAB program. The Ca2+ fluorescence emission intensity was expressed as F/F0 where the F0 was the basal fluorescence intensity level. The recording was performed at 35 °C.[Ca1/3)39.The extracellular FP produced by hESC-CMs was measured by multi-electrode array (MEA) assay using Multi Channel Systems MCS GmbH . Clusters of contracting hESC-CMs were plated on Matrigel® coated MEA chips containing 59 titanium electrodes and 1 internal reference electrode. Stocks of CGs were diluted to various concentrations in cell cultural medium . Data was acquired with an interval of 5 minutes at baseline and post drug applications. The FPD was analysed and corrected by the beating frequency (FPDc) with Fridericia’s formula , which approved this study. Developmental stages are in hours post fertilization (hpf) at 28.5 °C41.Wild-type zebrafish  were maintained as described4.7H20 1.2, Ca(NO3)2.4H2O 1.8, HEPES 15, adjusted to pH 7.2. The micropipette tip was positioned right above the pericardial region of the zebrafish heart. The electrical signals were recorded by pCLAMP 10 software (Molecular Devices) after amplification via Multiclamp 700B amplifier  and digitization through Axon Digidata 1440A digitizer (Molecular Devices). Data were analysed with Clampfit 10 software (Molecular Devices). For controls, the zebrafish larvae were mounted  in 1% low melting agarose in a glass dish and submerged in external buffer: 1X Egg water . To assess the effect of drugs on the heart activity of zebrafish, the mounted larvae are pre-treated with the various drugs (diluted in1X Egg water). APDs were determined by measuring the adjacent peaks of the action potentials using the pClamp software. APDs were corrected by heart rate as above-mentioned.Micropipettes for AP measurement on whole zebrafish larvae were prepared by pulling fire-polished borosilicate glass capillaries (World Precision Instruments) using the Flaming/brown micropipette puller P-1000 (Sutter Instrument). The micropipette was filled with internal buffer contained (in mM): NaCl 174, KCl 2.1, MgSO26 and Luo-Rody (LRd) model (1991) of mammalian (Guinea-pig) ventricular myocytes27 were adopted. The cycle length was 1 s.O’Hara-Rudy (ORd) model (2011) of non-diseased human midmyocardial ventricular myocytesp-value of <0.05 was considered statistically significant.Numerical data are presented as mean ± standard division (SD) or mean ± standard error of mean (SEM). Comparisons were made with paired and unpaired (two-tailed) Student t-test, One-Way repeated measures ANOVA followed by the Tukey’s post hoc testing and Two-Way repeated measures ANOVA followed by the Bonferroni post hoc testing using GraphPad Prism 5.0 . A


A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants.Ca2+‐responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress.We used single‐cell imaging of Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo‐osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo‐osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol.We found that salt stress induced a single Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.Our results indicate that the requirement for Ca In plants, calcium  which are sensed by a range of downstream effector proteins , calmodulin‐like proteins (CMLs), the calcium‐dependent protein kinases (CDPKs) or the calcineurin B‐like (CBL) calcium sensor proteins and their CBL‐interacting protein kinases (CIPKs)) , the transient receptor potential (TRP) channels and the inositol triphosphate receptor (IP3R)  . Voltage‐gated Ca2+ channels in the flagella are activated by a whole‐cell depolarization mediated by channelrhodopsin, a light‐gated ion channel situated in the plasma membrane adjacent to the eyespot  in gliding C. reinhardtii cells indicates the potential for complex and highly dynamic signalling, with each individual flagellum capable of generating very rapid, repetitive [Ca2+]fla elevations independently of the cytosol and the other flagellum  and cw15  were obtained from the Chlamydomonas Resource Center  and the Culture Collection of Algae and Protozoa , respectively. The ift20 IFT20‐mCherry strain was a gift from Karl Lechtreck  (10 000 MW) and the reference dye Texas Red Dextran (TR) (10 000 MW) . Dextran‐conjugated dyes were used to avoid the problems with dye compartmentalization commonly found with plant and algal cells  microscopy. Chlamydomonas reinhardtii cells were concentrated by centrifugation (400 g for 5 min) and washed with biolistic loading buffer (BLB) , and then biolistic loading was performed using a Bio‐Rad PDS‐1000 delivery system with 1100‐psi rupture discs, as described previously . The dyes demonstrated a homogenous cytosolic localization, with no evidence of compartmentalization. Chlorophyll autofluorescence was negligible relative to OG, allowing us to subsequently use epifluorescent microscopy to image [Ca2+]cyt.Cells were simultaneously loaded with two fluorescent dyes, the CaChlamydomonas Assay Buffer (CAB) containing 5 mM HEPES, 1 mM HCl, 1 mM KCl, 0.2 mM Ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid and 0.5 mM CaCl2 with pH adjusted to 7.4 using N‐methyl‐d‐glucamine (NMDG). Free Ca2+ was calculated to be 301 μM using Maxchelator (http://maxchelator.stanford.edu/). Cells were placed in 35‐mm glass‐bottomed dishes  coated with 0.01% poly‐L‐lysine  to encourage adherence of the cells. Cells were perfused with CAB buffer at a flow rate of 3 ml min−1 during imaging.Before imaging, cells were resuspended in nis‐elements v.3.1 software  with a 200–300‐ms exposure (frame rate of 3.33–5 frames s−1). The same microscopy equipment was used in TIRF mode for flagellar imaging. IFT20‐mCherry was visualized in TIRF mode using 561‐nm laser excitation and 575–625‐nm emission.Cells were imaged by epifluorescence microscopy at room temperature using a Nikon Eclipse Ti with a ×100, 1.49 NA oil immersion objective and detection with a Photometrics Evolve EM‐CCD camera . Simultaneous excitation of OG and TR was performed using 488‐ and 561‐nm lasers . A Dual‐View beam splitter device  was used to detect emission at 500–550 and 575–625 nm, respectively. Images were captured using 2. To investigate the response to hypo‐osmotic shock in the absence of contractile vacuole activity, cells were equilibrated to CAB containing 100 mM sucrose for at least 1 h. In order to assess whether extracellular Ca2+ is involved in [Ca2+]cyt elevations in response to osmotic shock, cells were initially perfused with Ca2+‐free CAB (containing 200 μM EGTA without added CaCl2) and a hypo‐osmotic shock was administered using deionized water containing 50 μM EGTA (adjusted to pH 7). In order to assess the involvement of mechanosensitive ion channels in the generation of [Ca2+]cyt elevations, cells were pretreated for at least 10 min with a final concentration of either 5 μM GsMTx4  or 10 μM Ruthenium red (RuR) (Sigma‐Aldrich) before the perfusion with distilled water or distilled water containing 10 μM RuR, respectively. RuR did not exhibit any fluorescent properties that interfered with Ca2+ imaging.NaCl shocks were delivered by rapidly switching the perfusion from CAB to CAB + 50–400 mM NaCl. Hypo‐osmotic treatments were delivered by switching the perfusion to deionized water containing 50 μM CaCl2+]cyt were identified by calculating the fluorescence intensity ratio between OG and TR for a defined region of interest within each C. reinhardtii cell. Ratio traces were smoothed using a Savitsky–Golay filter  and a baseline trace was created using an asymmetric least squares smoothing baseline algorithm  . The smoothed OG/TR traces were divided by the baseline trace to determine the relative change in fluorescence. [Ca2+]cyt elevations were defined as any increase in OG/TR fluorescence above a threshold value (3%) that persisted for more than one frame. We calculated that an OG/TR ≥ 3% required a minimum signal to noise ratio (SNR) of 10, with SNR defined as signal intensity/SD of noise. Contractile vacuole activity was detected by measuring changes in the fluorescence intensity of TR in a region of interest encompassing each contractile vacuole. For visualization of [Ca2+]cyt dynamics in cells, imagej software was used to divide each OG image by a rolling median image from 20 frames to produce a pseudocoloured ratio image. Previous researchers have demonstrated that standard Ca2+ ionophores do not work well in C. reinhardtii , allowing the increase in [Ca2+]cyt for a given change in fluorescence to be estimated, assuming a resting [Ca2+]cyt of 100 nM . Data obtained from all treatments are shown as mean ± SE.Changes in cyt elevations in plants to C. reinhardtii strain CC1201. We found that hydrogen peroxide (up to 20 mM) and mannitol (up to 400 mM) did not induce [Ca2+]cyt elevations in C. reinhardtii , suggesting that a threshold stimulus is required to trigger a [Ca2+]cyt elevation. Very few [Ca2+]cyt elevations were observed at lower concentrations of NaCl (no cells responded at 150 mM (n = 11) and only one out of 12 cells at 200 mM NaCl). Higher concentrations of NaCl (400 mM) shrank the cells excessively, preventing accurate determination of [Ca2+]cyt. Previous researchers have demonstrated that Ca2+‐dependent signalling pathways in C. reinhardtii can be influenced by the concentration of external Ca2+ in the surrounding media .In order to examine whether Ca2+ signalling in the cell wall‐deficient strain cw15 was significantly more sensitive to salinity stress than in CC1021. We found that 50 mM NaCl induced a single [Ca2+]cyt elevation in 55% of cw15 cells (n = 14), with a mean maximal increase in the OG : TR ratio of 8.0 ± 0.9% (176 nM [Ca2+]cyt) which occurred 14.4 ± 1.5 s after the application of the stimulus  resulted in progressive increases in the mean maximal amplitude of the [Ca2+]cyt elevations and in the proportion of cells exhibiting [Ca2+]cyt elevations  and occurred 4.7 ± 0.4 s after the application of the stimulus. We conclude that the timing and amplitude of NaCl‐induced [Ca2+]cyt elevations are sensitive to the intensity of the stimulus, which may contribute to specificity in downstream responses.Ca2+]cyt elevations induced by NaCl. We found that the [Ca2+]cyt elevations induced by 300 mM NaCl in CC1021 originated in the apex of the cell and rapidly spread to the remainder of the cell, representing a fast Ca2+ wave (n = 5 cells examined where the apex of the cell could be clearly distinguished) . Chlamydomonas reinhardtii is a small cell (5–10 μm), making it difficult to accurately determine the speed of the propagating Ca2+ wave. However, it was clear that the initial apical [Ca2+]cyt elevation spread to the remainder of the cell within 0.5–1 s, representing a speed of c. 10–20 μm s−1. We have previously observed that 20 mM external Ca2+ can stimulate a Ca2+ wave that propagates from the apex of C. reinhardtii cells at a mean speed of 30 μm s−1  from the endoplasmic reticulum . We did not observe [Ca2+]cyt elevations in any cells (n = 16). This indicates that an influx of external Ca2+ is required to initiate the [Ca2+]cyt elevations induced by NaCl, although the [Ca2+]cyt elevations may subsequently propagate through the release of Ca2+ from internal stores. The absolute dependence on external Ca2+ in C. reinhardtii contrasts with Arabidopsis, where NaCl‐induced [Ca2+]cyt elevations were only partially repressed in the presence of La3+ or EGTA  to expel excess water  routinely induced a series of repetitive [Ca2+]cyt elevations . The mean number of [Ca2+]cyt elevations observed within 60 s of hypo‐osmotic shock was 3.93 ± 0.47 and the average duration of each [Ca2+]cyt elevation was 1.91 ± 0.50 s . The mean maximal amplitude of the [Ca2+]cyt elevations was 6.08 ± 0.50%, equating to 166 nM [Ca2+]cyt (n = 14 cells). The initial [Ca2+]cyt elevation occurred 15.93 ± 1.53 s after the stimulus was applied. In contrast to [Ca2+]cyt elevations induced by NaCl, the [Ca2+]cyt elevations induced by hypo‐osmotic shock did not originate in the apex and appeared to be uniformly distributed throughout the cytosol . To investigate this further, we examined the response to hypo‐osmotic shock in two further C. reinhardtii strains. We did not observe any [Ca2+]cyt elevations in another cell wall‐deficient strain CC3395 (n = 11 cells), but found that 53.9% of CC125 cells  exhibited [Ca2+]cyt elevations (n = 13).Hypo‐osmotic stress causes a rapid influx of water into algal cells and may lead to catastrophic cell bursting if the cell cannot rapidly osmoregulate  provided a robust and reproducible stimulus that enabled us to study the nature of Ca2+ signalling in C. reinhardtii. However, the ionic compositions of the pretreatment (CAB) and treatment solutions are not identical and it is possible that changes in pH, Ca2+ or other ions also contributed to the observed [Ca2+]cyt elevations in addition to the change in osmolarity. To examine this possibility, we applied additional hypo‐osmotic stimuli to C. reinhardtii cells. We did not observe [Ca2+]cyt elevations when cells acclimated to CAB + 50 mM sorbitol were returned to CAB (n = 7 cells). However, cells acclimated to CAB + 100 mM sucrose gave robust and reproducible [Ca2+]cyt elevations when switched to 10% CAB (adjusted to 300 μM Ca2+ and pH 7.4) (seven out of 11 cells examined). This suggests that the [Ca2+]cyt elevations observed were not attributable to changes in Ca2+ or pH. While we cannot rule out a contribution from other ions (e.g. Cl−), we conclude that the rapid switch to a solution of very low osmolarity is likely to be the primary contributor to the observed [Ca2+]cyt elevations. The initial hypo‐osmotic stimulus (deionized water + 50 μM CaCl2) was used for all further experiments.The hypo‐osmotic stress applied  . Five‐micromolar GsMTx4 resulted in a lower proportion of cells exhibiting [Ca2+]cyt elevations  and these cells only exhibited single rather than repetitive [Ca2+]cyt elevations. GsMTx4 did not have any effect on the timing and the duration of the initial [Ca2+]cyt elevation.The response to hypo‐osmotic shock in CC1021 cells was dependent on the presence of external Ca21) Fig. a. Our re2+‐dependent signalling pathways have been implicated in the regulation of CV activity in several unicellular protists  events of the CV. Furthermore, we found no evidence to suggest that Ca2+ signalling influenced the frequency of systole events during hypo‐osmotic shock. The mean time between systole events was 21.8 ± 1.0 s in control cells and was 17.8 ± 0.7 s during hypo‐osmotic shock (n = 13 cells). In Ca2+‐free media, the mean time between systole events was 21.4 ± 1.2 s in untreated control cells and 18.4 ± 0.4 s during hypo‐osmotic shock (n = 18 cells). In cells treated with 10 μM RuR, the mean time between systole events was 20.6 ± 1.2 s before and 20.5 ± 1.1 s during hypo‐osmotic shock (n = 17 cells). The timing of systole events between the treatments was not statistically significant . To examine whether the [Ca2+]cyt elevations could be generated in the absence of CV activity, we acclimated C. reinhardtii cells to mildly hypertonic medium (CAB + 100 mM sucrose) to minimize CV activity  and these were primarily restricted to the period before the onset of full CV activity . The remaining cells did not exhibit [Ca2+]cyt elevations, but still showed full restoration of CV activity. Thus, the [Ca2+]cyt elevations induced by hypo‐osmotic shock are not dependent on CV activity and are not required for the initiation of CV activity.CaChlamydomonas reinhardtii cells have two flagella c. 10 μm in length that are used for swimming and gliding motility. Although the two flagella represent only 0.27% of the total cell volume (assuming a cell radius of c. 5 μm), they constitute a much larger proportion of the cell surface area (c. 7.2%) and may therefore play an important role in osmotic signalling responses. Certain osmotic stimuli have previously been shown to induce deflagellation. For example, application of 50 mM KCl to Chlamydomonas moeuwsii caused rapid deflagellation (within 30 s), whereas higher concentrations of KCl caused a much slower deflagellation response  microscopy, we found that salinity stress induced by 300 mM NaCl for 30 s did not result in deflagellation in CC1021 cells (zero out of 43 cells deflagellated). Applying a hypo‐osmotic stress for 30 s also did not induce deflagellation (0 out of 50 cells deflagellated). Examination of cells loaded with Ca2+‐responsive dyes confirmed that deflagellation does not occur during [Ca2+]cyt elevations induced by hypo‐osmotic shock . Therefore, the observed [Ca2+]cyt elevations induced by these stressors are not linked to the deflagellation process.2+ elevations in flagella and whether these were related to the observed [Ca2+]cyt elevations. We have previously demonstrated that large [Ca2+]cyt elevations  can result in simultaneous [Ca2+]fla elevations in both flagella , with an average of 3.06 ± 0.27 Ca2+ elevations per flagellum . Furthermore, [Ca2+]fla elevations were observed in cells that did not demonstrate [Ca2+]cyt elevations. The results suggest that the flagella and the cytosol can act as independent Ca2+ signalling compartments in response to hypo‐osmotic shock.We found that the rapid fla elevations observed during hypo‐osmotic shock influence flagellar‐mediated gliding motility through their action on the accumulation of IFT particles  in the endoplasmic reticulum, leading to a spatially propagating [Ca2+]cyt elevation. Fast Ca2+ waves have not been characterized in detail in individual cells of land plants, with the exception of a UV‐induced fast Ca2+ wave in Physcomitrella patens caulonema cells , a putative plasma membrane Ca2+‐permeable channel, is responsible for a Ca2+‐dependent Ca2+ influx in the presence of 220 mM NaCl  proteins, probably contribute to Ca2+ influx during hypo‐osmotic shock  family (Msy1 and Msy2) contribute to Ca2+ signalling responses of walled and wall‐less strains to osmotic stimuli. The wild‐type walled strain CC1021 was much less sensitive to NaCl than the wall‐less strain cw15, but conversely the wall‐less strains did not exhibit Ca2+ elevations in response to hypo‐osmotic stress. These observations suggest that the cell wall may contribute to osmotic signalling, although the underlying mechanisms are not yet clear. Interactions between the plasma membrane and the cell wall have been suggested to contribute to mechanical stimuli during both hypo‐ and hyperosmotic stress in Fucus rhizoids  was found to cause flagellar shortening and deflagellation in C. reinhardtii  and do not exhibit dynamic changes in [Ca2+]  should be directed to the Fig. S1 Processing of data during Ca2+ imaging.Fig. S2 In vitro calibration of Oregon Green BAPTA dextran.Fig. S3 Spontaneous [Ca2+]cyt elevations induced by 10 mM external Ca2+.Fig. S4 [Ca2+]cyt elevations induced by hypo‐osmotic stress occur in the absence of contractile vacuole activity.Click here for additional data file.Video S1 A Ca2+ wave in Chlamydomonas reinhardtii induced by NaCl shock.Click here for additional data file.


N = 116). Women in PROGRESS attended an off-site weekend workshop and gained access to a network of volunteer female scientific mentors from on- and off-campus . The results indicate that women in PROGRESS had larger networks of developmental mentoring relationships and were more likely to be mentored by faculty members and peers than matched controls. Mentoring support from a faculty member benefited early-undergraduate women by strengthening their scientific identity and their interest in earth and environmental science career pathways. Further, support from a faculty mentor had a positive indirect impact on women’s scientific persistence intentions, through strengthened scientific identity development. These results imply that first- and second- year undergraduate women’s mentoring support networks can be enhanced through provision of protégé training and access to more senior women in the sciences willing to provide mentoring support.Women are underrepresented in a number of science, technology, engineering, and mathematics (STEM) disciplines. Limited diversity in the development of the STEM workforce has negative implications for scientific innovation, creativity, and social relevance. The current study reports the first-year results of the PROmoting Geoscience Research, Education, and SuccesS (PROGRESS) program, a novel theory-driven informal mentoring program aimed at supporting first- and second-year female STEM majors. Using a prospective, longitudinal, multi-site , propensity score matched design, we compare mentoring and persistence outcomes for women in and out of PROGRESS ( In the United States, women continue to be underrepresented in many science, technology, engineering, and mathematics (STEM) disciplines , includirole modeling support involves serving as an inspiration and example of success, as well as, being a guide for the norms, behaviors, and values that are needed to succeed ).Student motivation, operationalized as deep interest in earth and environmental sciences, was our first outcome variable. Students’ deep interest was assessed with a two-item measure adapted from prior literature on scientific interest development . ParticiStudent’s scientific persistence intentions was our second outcome variable. We measured scientific persistence intentions with a two-item measure adapted from prior literature on scientific persistence . Studentp’s > .05).All analyses were conducted in SPSS software version 23. Prior to assessing research questions, we conducted exploratory data analysis to identify potential outliers and assess the tenability assumptions for regression analysis, see We assessed the degree to which PROGRESS enhanced student’s network of developmental mentoring relationships in two complementary sets of analyses. First, we examined the proportion of students who reported receiving mentoring support from one or more sources . Consistent with our expectations, control group students were more likely to report having a single mentor compared to having multiple mentors, while PROGRESS students were more likely to reporting having multiple mentors compared to having a single mentor, see 2 = 5.94, p = .05, ϕ = .23.A chi-square test of independence , 71, comχ2(df = 1) = 0.36, p = .54, B = 0.38, Odds-ratio [O.R.] = 1.46, 95% CI ); however, the odds of having multiple mentors vs. one-mentor were over three-times higher in the PROGRESS group compared to the control group . These results indicate that the PROGRESS program enhanced the size of student’s networks of developmental mentoring relationships from one to multiple mentors.To fully describe the pattern of differences, we compared the odds of having one-mentor vs. no mentor and odds of having multiple mentors vs. one-mentor in the PROGRESS and control groups using hierarchical logistic regression analysis  of college campus to control for the nesting of students within schools) , 73. Theχ2[df = 1] = 6.87, p = .009, B = 1.25, O.R. = 3.48, 95% CI ). Analysis revealed a similar trend for having a peer mentor, but the trend did not exceed a conventional level of statistical significance . PROGRESS and control members had equally low rates of being mentored by graduate students  and scientific professionals outside of the university .To further test the impact of PROGRESS on mentoring support, we conducted a series four of hierarchical logistic regression models predicting each of the four sources of mentoring support  from PROGRESS membership, controlling for college campus. Consistent with our expectations, the odds of having a faculty mentor were 3.5-times higher for PROGRESS members than control participants  were highly correlated with the size of the student’s network of developmental mentoring network . Therefore, we chose to proceed using only the sources of mentoring support as a predictive variable, as these provide a better insight into how specific kinds of mentoring support enhance student identity, motivation, and persistence intentions. In addition, we tested the indirect effects of PROGRESS membership and mentoring on interest and persistence through scientific identity.In the first model, scientific identity was predicted from binary-coded indicators of the four sources of mentoring support  and PROGRESS membership, controlling for college campus (step-1). The analysis revealed that only faculty mentorship status predicted scientific identity, such that students with a faculty mentor had higher scientific identity scores compared to those without, see βa×b Partial Standardized = .41, ba×b = 0.44, bias corrected 95% CI ). Thus, participants with a faculty mentor had persistence intentions 0.41 standard deviations higher compared to those without due to the indirect effect of mentorship on scientific identity, see We tested for mediation by estimating the bootstrapped  bias corrected 95% confidence intervals around the indirect effect of faculty mentoring on persistence intentions through science identity using the PROCESS macro . The anaTaken together, the analyses indicate that support from a faculty mentor is associated with higher levels of scientific identity development and deeper interest in earth and environmental sciences. Furthermore, the conditional process analysis indicated that faculty support is associated with higher scientific persistence intentions, through scientific identity development.Female undergraduates can experience social barriers that undermine their scientific identity development, motivation, and persistence in STEM education and career pathways , 7. The Consistent with our expectations, undergraduate women in PROGRESS were more likely to have multiple mentors and were more likely to receive support from faculty and peer mentors compared to their matched female controls. These results are consistent with research showing that, in the absence of a deliberative mentoring intervention, most first-year students (in this case 48%) tend to seek out a single mentor . We wereTaken together, these findings extend the mentoring literature in several important ways. First, much of the research on mentoring undergraduates has focused on upper-division science majors in the context of multicomponent science training programs  , 23–27. Although the present study addressed several gaps in the mentoring literature, there are at least two distinct limitations to our inferences. First, these data only follow changes in college women’s experiences across a total of six months out of a single academic year. It is probable that the developmental needs of aspiring scientists change as they advance toward graduation and more advanced career pursuits. Therefore, we expect that the observed benefits of receiving mentoring support from a faculty member as well as the lack of observed benefits of receiving mentoring support from other sources will change over time. The focus on early tenure students and the relatively short timeframe limit our ability to examine dynamic changes in mentoring benefits in the different phases of undergraduate tenure and over time in this report. However, because the PROGRESS program is ongoing, future studies will examine the changing benefits of faculty and near-peer mentors for female science majors over a longer period of time.A second potential limitation concerns the design of the control group. The current study used a PSM approach to construct an “inactive” or treatment as usual control. Comparisons with an inactive control group allow for an assessment of the overall benefits of a treatment package, but do not allow inferences about individual components of a treatment package , 56. In Mentoring and psychological theories suggest that women in some scientific disciplines face unique challenges to persistence and benefit from having larger networks of developmental mentoring relationships. We examined the benefits a novel informal mentoring program (PROGRESS) aimed at supporting first- and second-year female STEM majors’ pursuit of scientific career pathways, with particular focus on careers in earth an environmental sciences.We found that PROGRESS expanded participants’ networks of developmental mentoring relationships compared to a matched control group. In particular, PROGRESS members were more likely to receive mentoring support from faculty members and peers compared to matched controls.Mentoring support from a faculty member strengthened participant’s scientific identity, motivation, and (indirectly) persistence intentions. These factors have been shown by prior studies to lead to longer-term persistence and achievement outcomes.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The endoplasmic reticulum (ER) is the main and largest intracellular Ca2+ store. At the ER two protein families of Ca2+ release channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), are expressed. Several studies have reported roles in the regulation of autophagy for the ubiquitously expressed IP3R. For instance, IP3R-mediated Ca2+ release supresses basal autophagic flux by promoting mitochondrial metabolism, while also promoting the rapid initial increase in autophagic flux in response to nutrient starvation. Insights into the contribution of RyRs in autophagy have been lagging significantly compared to the advances made for IP3Rs. This is rather surprising considering that RyRs are predominantly expressed in long-lived cells with specialized metabolic needs, such as neurons and muscle cells, in which autophagy plays important roles. In this review article, recent studies revealing roles for RyRs in the regulation of autophagy will be discussed. Several RyR-interacting proteins that have been established to modulate both RyR function and autophagy will also be highlighted. Finally, the involvement of RyRs in neurodegenerative diseases will be addressed. Inhibition of RyR channels has not only been shown to be beneficial for treating several of these diseases but also regulates autophagy.Intracellular Ca Within the lumen of the ER maintaining adequate Ca2+ levels is essential to promote the correct folding of proteins. Failure to do so results in ER stress and activation of the unfolded protein response  receptors  via calmodulin kinase kinase β leading to the inhibition of the mechanistic target of rapamycin (mTOR), thereby inducing autophagy  initiated at the level of the lysosomes via NAADP-sensitive Ca2+ channels. Pharmacologically RyRs can be activated by caffeine or low ryanodine concentrations (nM range), locking the channel in a sub-conductance state. Inhibition of RyRs can be obtained most specifically via a high ryanodine conentrations (>20 μM) or with dantrolene, a FDA-approved RyR blocker.RyRs are large (>500 kDa) ER-located Ca2+, Mg2+ and ATP), post translational modifications and protein interactions  domains Letai, . Anti-apL bind to and inhibit RyRs  1 and 2 are important regulators of intracellular Ca2+ oscillations in the cardiomyocytes and reduced the ER Ca2+ store content. Recently, it was shown that a decrease in PC2 expression increased the expression of RyR2 and SERCA2A in the heart while phosphorylated phospholamban, a SERCA inhibitor in its non-phosphorylated form, was decreased  is a progressive neurodegenerative disease of which several types are known to exist (Sun et al., 2+ release. It will be interesting to see whether these interactors also influence autophagy by modulating RyR-mediated Ca2+ release.RyR signaling and autophagy are two factors critically linked to the correct functioning and survival of neurons (Figure 2+ release and autophagy have also been shown to contribute to several neurodegenerative diseases. Disease causing mutant proteins, excitotoxicity and aging are not only the cause for these diseases but also change RyR function and autophagy. As RyR function and autophagy are involved in maintaining neuronal health, changes in these processes may result in aggravating disease progression (Figure 2+ release has been shown to confer beneficial effects in treating a number of these diseases. Several of the above mentioned studies used the FDA-approved drug dantrolene in order to block RyR-mediated Ca2+ release. An observation common in the studies looking at how RyRs regulate autophagy, is that RyR inhibition via dantrolene changes/normalizes autophagic flux. This may in part underlie the beneficial effects of dantrolene in several neurodegenerative diseases as by inhibiting RyR activity it may also regulate autophagic flux indirectly (Figure Alterations in RyR-mediated CaTV wrote this review article.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.