MICU1 is a regulatory subunit that shields mitochondria from Ca2+ overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca2+ uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca2+ uptake. Our data demonstrate that mitochondrial Ca2+ uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca2+ sensitivity. UCP2/3 normalize Ca2+ sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca2+ uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca2+ uniporter by PRMT1 and UCP2/3.Recent studies revealed that mitochondrial Ca MICU1 is a regulatory subunit of mitochondrial Ca2+ channels that shields mitochondria from Ca2+ overload. Here the authors show that MICU1 methylation by PRMT1 reduces Ca2+ sensitivity, which is normalized by UCP2/3, re-establishing mitochondrial Ca2+ uptake activity. By identifying mitochondrial Ca2+ uptake 1 (MICU1) as an essential component of mitochondrial Ca2+ uniport, Mootha and colleagues2+ uptake2+ uptake machinery including the mitochondrial uniporter MCU27892+ uptake is accomplished by a sophisticated heteromeric protein complex111213151017202+ uptake. However, the molecular mechanisms how these proteins actually add to or influence MCU activity remain largely unresolved and the direct regulation of mitochondrial Ca2+ uptake by these proteins has been questioned recently14232+ uptake machineries in different cell types. Notably, UCP2/3 were found to be fundamental for efficient mitochondrial Ca2+ uptake in all our related studies22252728292+ current in mitoplasts isolated from HeLa cells2+ signalling was found20332+ uptake.The identity of mitochondrial Ca2+ uniporter complex. In particular, protein arginine methyl transferase 1 (PRMT1)-mediated methylation of MICU1 desensitizes this protein for Ca2+ and, in turn, reduces mitochondrial Ca2+ uptake. UCP2 binds exclusively to the methylated form of MICU1, normalizes its Ca2+ sensitivity and re-establishes mitochondrial Ca2+ uptake. Thus, UCP2/3 are described as sensitizer of mitochondrial Ca2+ uptake under conditions of increased PRMT1 activity, a phenomenon that has probably great importance in cancer cells.Here we show that posttranslational protein modification of MICU1 regulates the activity of the mitochondrial Ca2+ uptake. As previously reported, small interfering RNA (siRNA)-mediated knockdown of UCP2/3 yielded strong reduction of mitochondrial Ca2+ uptake on intracellular Ca2+ release by any IP3-generating agonist in HeLa cells  Ca2+ ATPase activity, and activity of mitochondrial Na+/Ca2+ exchangerFirst, we searched for cell types with and without an obvious role of UCP2/3 in mitochondrial CaLa cells  and Ea.hLa cells , a hybrinditions , althougnditions . Howeverll types , confirm2+ uptake to UCP2/3 knockdown in these cell types of same origin was surprising and offered us the possibility to further explore the principles that are responsible for an engagement of UCP2/3 in mitochondrial Ca2+ uptake. In view of the various and inconclusive messenger RNA expression patterns of some constituents of the MCU complex in all cell types used  from UCP2/3 and/or PRMT1. The Ca2+ sensitivity of mitochondrial Ca2+ uptake decreased in PRMT1-active wild-type HeLa cells from 5.3 (4.1–6.7) μM in controls to 14.1 (12.0–16.5) μM in cells that were depleted from UCP2/3  μM Ca2+; without UCP2/3: EC50=4.7 (3.5–6.3) μM Ca2+; 2+ uptake machinery.To understand how UCP2/3 contribute to mitochondrial Cam UCP2/3 , indicat2+ uniporter complex to allow Ca2+ influx into the mitochondrial matrix essentially requires MCU22+ to the EF-hand domains at the carboxy-terminal end of MICU1  or phenylalanine (MICU1-F). Both MICU1 mutants could not be methylated by PRMT1 (2+ levels compared with cells overexpressing wild-type MICU1 (97–476 and MICU197–444). Investigation of the asymmetric protein arginine methylation status with the aDMA antibody demonstrated that only the long version, containing arginine at position 455, could be methylated by PRMT1 . We found that methylation of MICU1 strongly reduced its sensitivity for Ca2+ to trigger de-oligomerization from a dissociation constant (Kd) of 5.4±0.7 μM for non-methylated MICU1 to 14.0±1.5 μM binding of Ca2+ to methylated MICU1 , indicating that R455 is the only residue modified by PRMT1 2+ sensitivity towards a 2.6-fold higher Ca2+level.Analysis of the published crystal structure of MICU1 revealed that R455 is localized in the C terminus , a regioed MICU1 . This deby PRMT1 . Notably2+-induced rearrangement of methylated MICU1, we tested for binding of UCP2 to methylated and non-methylated MICU1 using ITC. To avoid improper folding of the suspended membrane protein UCP2 in a liquid phase, we decided to use the chemically synthetized intermembrane loop 2 of UCP2 (IML2UCP2) that has been described to be essential for an engagement of UCP2/3 to mitochondrial Ca2+ uptake22UCP2 to purified non-methylated and methylated MICU1 and its impact on Ca2+ sensitivity, were tested in vitro using ITC. These experiments unveiled that the IML2UCP2 exclusively binds to methylated MICU1 with a Kd of 24.6±17.7 μM (UCP2 to non-methylated MICU1 was detected (UCP2 had no impact on the Ca2+ sensitivity of non-methylated MICU1 (14.7±6.4), it shifted the Ca2+ sensitivity of methylated MICU1 (17.1±9.9) towards that of the non-methylated MICU1  imaging in HeLa cells co-expressing MICU1 fused to either cyan fluorescent protein (MICU1-CFP) or yellow fluorescent protein (MICU1-YFP) as previously described2+-induced rearrangement of MICU1 multimers in HeLa cells, an EC50 of 4.0 (3.3–5.0) μM was obtained (2+ (EC50=3.5 (2.6–4.6) μM; 2+ sensitivity of MICU1 and yielded a 4.6-fold increase in the EC50 of Ca2+-triggered MICU1 rearrangement to 18.5 (14.3–23.8) μM (2+ sensitivity of MICU1 rearrangement (EC50=3.8 (3.0–4.8) μM; 2+ sensitivity of the non-methylated MICU1-K expressed in HeLa cells was independent from the presence of UCP2/3 (with UCP2/3: EC50=2.1 (1.6–2.9) μM Ca2+; UCP2/3 knockdown EC50=3.0 (2.3–3.9) μM Ca2+  might be due to the fact that purified proteins were measured in solution and not embedded in membranes. Nevertheless, these data emphasize that the Ca2+-dependent activation of mitochondrial Ca2+ uniport, which is under the control of methylated MICU1, requires much higher Ca2+ levels and exhibits less activity on identical Ca2+ elevations  μM . Importa μM Ca2+ . These devations . However2+ in two cancer-type cell lines HeLa and Ea.hy926  but could introduce UCP2/3 dependence of MCU activity in short-term cultured HUVEC cells by PRMT1, we speculate that the contribution of UCP2/3 in mitochondrial Ca2+ uptake rather depends on PRMT1 activity than whether or not the cell is immortalized. Nevertheless, as cancer is often associated with enhanced PRMT activity2+ uptake.Although several core proteins of the mitochondrial uniporter complex could be identified so far   of the MCU complex also serves as target of PRMT1, our data highlight methylation of MICU1 as being the essential step for the engagement of UCP2/3 in the regulation of mitochondrial Ca2+ uptake.Interestingly, we found an exclusive methylation of MICU1 but not of MCU and UCP2. Notably, methylation of MICU1 by PRMT1 was independent from the expression of UCP2/3. Thus, a direct effect of UCP2/3 on PRMT1 activity or MICU1 methylation appears unlikely. Therefore, we concentrated on MICU1 as the most important gatekeeper of the MCU complex15CU1 refs , 44. Thi2+ elevation, which results in binding of Ca2+ to the EF-hand domains of MICU1, triggers a clear rearrangement of MICU1 multimers independently from the mitochondrial matrix Ca2+ concentration, mitochondrial membrane potential, and the level of expression of either MCU or EMRE2+-induced rearrangement of MICU1 proteins is, however, essential to allow an efficient mitochondrial Ca2+ uptake via the MCU/EMRE channel. In line with the reduction of sensitivity of the mitochondrial Ca2+ uptake by PRMT1-mediated methylation of MICU1, in vitro ITC revealed a strongly reduced sensitivity for Ca2+ to trigger de-oligomerization of MICU1 on methylation of MICU1 at position 455. Excitingly, the range of Ca2+ desensitization of MICU1 oligomerization by methylation in the ITC measurements perfectly matches that on mitochondrial Ca2+ uptake and of the Ca2+-induced MICU1 rearrangement. Hence, as mutation at position 455 of the MICU1 prevented Ca2+ desensitization in the ITC measurements as well as in our MICU1 FRET experiments, we conclude that PRMT1-mediated methylation of the arginine at position 455 of MICU1 is responsible for the loss of Ca2+ sensitivity of MICU1.Very recently we demonstrated that a cytosolic/intermembrane Ca2+-induced oligomerization of methylated MICU1, while UCP2 did not bind to non-methylated MICU1. These findings are in line with our data obtained in the MICU1–MICU1 FRET experiments where UCP2 re-established normal Ca2+ sensitivity of the Ca2+-induced rearrangement of methylated MICU1, whereas UCP2 had no effect on the rearrangement of non-methylated MICU1.Notably, our ITC experiments further demonstrate that UCP2 normalizes the reduced Ca2+ uptake and prove UCP2/3 as essential sensitizer of methylated MICU1 and, thus, being fundamental for establishing MCU activity under conditions where PRMT1 is upregulated  metabolism. So far, little is known on the modulation of the activity of the MCU complex. On the transcriptional level, the expression patterns of the proteins involved in the mitochondrial Ca2+ uptake were described to shape the activity of neuronal cell types572+-CREB pathway in chicken DT40 B lymphocytes162+ uptake are so far unknown but might be due to differences in medium compositions, growth rates, age, or passage number of the cell (line) used and/or cell handling before experiments. However, tissue-specific variations in PRMT1 activity2+ uniporter between various tissues502+ uptake/activity would counteract the elevated demand of cancer cells on Ca2+-triggered ATP production in the mitochondria. By using UCP2/3, cancer cells may be able to counteract the impact of an essential PRMT1 activity to mitochondrial Ca2+ uptake and, thus, re-establish activity of mitochondrial Ca2+ uptake to achieve Ca2+-triggered activation of ATP production. Notably, increased UCP2 expression has been reported in several cancer types502+ uptake fueling the hypothesis that UCP2 might play an important role in the energy balance of certain cancer cells. Obviously, further studies are required to explore the interrelation between UCP2/3 expression and PRMT1 activity in cancer and to reveal whether or not this function of UCP2/3 is causally linked to altered UCP2/3 expression levels in various cancer cells.Mitochondrial Ca2+ uptake machinery is under the control of posttranslational protein modification by PRMT1 that methylates MICU1 at position 455. Methylation of MICU1 yields reduced Ca2 sensitivity for protein rearrangement and, thus, reduced mitochondrial Ca2+ uptake. UCP2 is described as unique regulator of methylated MICU1 that normalizes the Ca2+ sensitivity of methylated MICU1 and, consequently, allows the re-establishment of mitochondrial Ca2+ uptake. Accordingly, although originally not involved in the regulation of mitochondrial Ca2+ uptake, UCP2/3 get engaged as sensitizer of methylated MICU1 preserving the activity of mitochondrial Ca2+ uptake despite the reduced Ca2+ sensitivity of methylated MICU1 . Histamine, BHQ, and EGTA were purchased from Sigma-Aldrich . Before experiments, cells were washed and maintained for 20 min in a HEPES-buffered solution containing 138 mM NaCl, 5 mM KCl, 2 mM CaCl−1 penicillin and 100 μg ml−1 streptomycin, and 2 mM glutamine . By routine, long-term cultured cells were tested for mycoplasma contamination and were tested to be free of mycoplasma. For Ca2+ imaging, cells were plated on 30 mm glass coverslips and transiently transfected at 60–80% confluence with 1.5 μg plasmid DNA encoding the appropriate sensor alone or with 100 μM siRNA using 2.5 μl of TransFast transfection reagent  in 1 ml of serum- and antibiotic-free medium. Cells were maintained in a humidified incubator  for 16–20 h. Afterwards, transfection mix was replaced by full culture medium. All experiments were performed 48 h after transfection. siRNAs were obtained from Microsynth  and their sequences (5′–3′) were as follows: human UCP2 siRNA: 5′- GCA CCG UCA AUG CCU ACA A dTdT-3′; human UCP3 siRNA: 5′- GGA ACU UUG CCC AAC AUC A dTdT-3′; human PRMT1 siRNA: 5′- CGU CAA AGC CAA CAA GUU A dTdT-3′; human MCU siRNA-1: 5′- GCC AGA GAC AGA CAA UAC U dTdT-3′; human MCU siRNA-2: 5′- GGA AAG GGA GCU UAU UGA A dTdT-3′; UCP2 siRNA: 5′- UGU CGC UCG UAA UGC CAU U dTdT-3′; human PRMT2 siRNA: 5′- CCC UGA CGG AUA AAG UCA U dTdT-3′; human PRMT3 siRNA: 5′- GCC UUG UAG CAG UGA GUG A dTdT-3′; human PRMT4 siRNA: 5′- GUA CAC GGU GAA CUU CUU A dTdT-3′; and human PRMT6 siRNA: 5′- CGG GAC CAG CUG UAC UAC G dTdT-3′. For detection primers, see HeLa S3 (ATCC CCL-2.2) and Ea.hy926 cells  were grown in DMEM medium (Sigma-Aldrich) containing 10% fetal bovine serum (PAA Laboratories), 100 U mlL-lysine-coated 30 mm glass coverslips in six-well plates, transiently transfected at 60–80% confluence with 100 μM siRNA using 2.5 μl of TransFast transfection reagent in 1 ml of serum- and antibiotic-free medium, and infected with BacMam 4mtD3cpv virus  following the CellLight protocol. Endothelial cells have been authenticated by factor VII and/or endothelial nitric oxide synthase (eNOS) staining. Experiments were performed 48 h after transfection and infection.HUVEC cells were isolated from umbilical vein by scraping and afterwards cultured in endothelial cell growth medium (EGM-2) . PAECs were harvested by scraping and cultured in DMEM containing supplements mentioned above. For imaging experiments, HUVEC cells and PAECs were seeded on poly-2+ uptake machinery and PRMT1 in HeLa and Ea.hy926 cells, as well as in HUVECs and PAECs, was examined by RT–PCR. A QuantiFast SYBR Green RT–PCR kit  was used to perform real time PCR on a LightCycler 480  and data were analysed by the REST Software (Qiagen). Relative expression of specific genes was normalized to human  or porcine GAPDH  and reverse transcription was performed in a thermal cycler (Peqlab) using a complementary DNA synthesis kit . Expression of core constituents of mitochondrial Cane GAPDH  respectine GAPDH  for realFor detecting the presence of the different PRMT isoforms in HeLa S3, Ea.hy926, HUVEC and in PAEC cells, specific primers (Invitrogen) were designed  and used2+]mito and [Ca2+]ER were followed in cells expressing the 4mtD3cpv or D1ER2, 1 mM MgCl2, 10 mM Hepes, 2.6 mM NaHCO3, 440 μM KH2PO4, 340 μM Na2HPO4, 10 mM D-glucose, 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin–streptomycin, pH adjusted to 7.4. Single-cell measurements were performed on a Zeiss AxioVert inverted microscope  equipped with a polychromator illumination system  and a thermoelectric-cooled CCD (charge-coupled device) camera . Transfected cells were imaged with a × 40 oil-immersion objective (Zeiss). Excitation of the fluorophores was at 440±10 nm  and emission was recorded at 480 and 535 nm using emission filters  mounted on a Ludl filterwheel. Devices were controlled and data were acquired by VisiView 2.0.3 (Visitron Systems) software and analysed with GraphPad Prism version 5.00 for Windows . Results of FRET measurements are shown as (Ri−Background)+[(Ri−Background)−(R0−Background)]  to correct for photobleaching and/or photochromism.Dynamic changes in [CaKd) of MICU1 FRET rearrangement were determined in diverse knockdown and overexpression experiments by the application of various Ca2+ concentrations ranging from 0.1 to 1,000 μM, to permeablized HeLa cells.MICU1 FRET measurements were performed as described previouslyF380/F340.Cells were incubated with 2 μM Fura-2/AM  in loading buffer for 30 min and were alternately illuminated at 340 and 380 nm, whereas fluorescence emission was recorded at 510 nmXXXAGTGAAACCCAT -3′, whereas XXX for MICU1-K is TTT and for MICU1-F AAA. The PCR fragment was cloned into a pcDNA3.1 (+) vector via BamHI and EcoRI sites already C-terminally containing the sequence coding for the red fluorescent protein mCherry. MICU1-CFP and MICU1-YFP were constructed by amplifying the coding sequences of human MICU1  without the stop codon from a HeLa cDNA by PCR. The following primers (Invitrogen) were used: forward: 5′- ACGGATCCACCATGTTTCGTCTGAACTCAC -3′ and reverse: 5′- ACGAATTCCTGTTTGGGTAAAGCGAAGTCC -3′. Subsequently, the PCR fragment was cloned into pcDNA3.1 (+) vector using BamHI and EcoRI sites. The coding sequences of enhanced CFP and citrine (YFP) were amplified with the same primers: forward: 5′- AAGAATTCATGGTGAGCAAGGGCGAGGAG -3′ and reverse: 5′- CCTCTAGAACTTGTACAGCTCGTCCATGC -3′. Each PCR product was C-terminally fused to hMICU1 using the EcoRI and XbaI sitesFor engineering MICU1 mutants (MICU1-K and MICU1-F), an already existing MICU1 plasmid was amplified via PCR with primers containing the respective mutation sequence. For both mutants the forward primer 5′- ACGGATCCATGTTTCGTCTGAACTCAC -3′ with a BamHI restriction site was used. As the mutations are very close to the end of MICU1, we decided to take a long reverse primer with a EcoRI restriction site and the following sequence: 5′- AAGAATTCCTGTTTGGGTAAAGCGAAGTCCCAGGCAGTTTCCTGTGCACATTTCCACATGGCCTGCATGAGCells were lysed with RIPA buffer (Biorad formulation), scraped, and sonicated . Protein A/G plus agarose beads  were washed twice with 1 ml IP washing buffer (Abcam formulation) and incubated with 1 μg anti-flag antibody  for 2 h at 4 °C. Protein lysates were added and incubated with the coated beads overnight at 4 °C on a shaker. After three washing cycles with 1 ml IP-washing buffer NuPAGE LDS sample buffer  and 1 μl of NuPAGE Sample Reducing Agent (Fisher Scientific) was added to the beads-lysate mix, heated for 10 min at 70 °C and 800 r.p.m. and, afterwards, directly loaded onto an SDS–PAGE gel.Western blots were performed following standard protocols. Briefly, samples were heat denatured in 1 × Laemmli sample buffer and resolved in an 10% SDS–PAGE gel and Mini-PROTEAN TGX precast gels  in parallel to PageRulerTM Plus Prestained Protein Ladder (Fisher Scientific) and then transferred onto a polyvinylidene difluoride membrane . To exclude unspecific bands due to impurities of immunoprecipitation, normal mouse IgG  and a control lysate  were subjected to western blotting. Proteins were detected using the following antibodies: MICU1 , MCU , UCP2 , Flag  and β-actin . The methylation status was detected by the asymmetric dimethyl arginine motif aDMA antibody  and the anti-mono and dimethyl arginine antibody (MMA/SDMA) . The secondary horseradish peroxidase antibodies, anti-mouse, anti-rabbit , anti-goat , and VeriBlot for IP antibody , were used and visualized with super signal west pico luminol/enhancer developing solution (Fisher Scientific). As a control for the IgG heavy and light chains, mouse normal IgG  was used.Hela cells were transfected with D1ER and PRMT1 construct or siRNA against PRMT1. After 2 days, cells were stained for 10 min with 200 nM MitoTracker Red CMXRos and imaged directly with 488 nm laser excitation (D1ER) and a 561 nm laser (MitoTracker Red CMXRos) with a CFI SR Apochromat TIRF × 100 oil (NA1.49) objective mounted on a Nikon-Structured Illumination Microscopy (SIM) System equipped with an Andor iXon3 EMCCD camera. Three-dimensional (3D) SIM was used in both channels with 30 ms exposure time per image. SIM images where reconstructed using Nis-Elements (Nikon). Images were background corrected with an ImageJ-Plugin . Further, the ImageJ/Fiji tool coloc2 was used to determine Pearson and Mander's coefficients, whereby images were thresholded with Costes automatic threshold to determine Mander's coefficient (overlap of mitochondria with ER).Hela cells were transfected with tagged MICU1 constructs (MICU1-WT/MICU1-K/MICU1-F). After 2 days, cells were stained for 10 min with 200 nM MitoTracker Red CMXRos and imaged directly. High-resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss) equipped with × 100 objective lens , a motorized filter wheel  on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems), and a Nipkow-based confocal scanning unit . The MICU1-YFP constructs and Mitotracker Red CMXRos were excited with 488 and 561 laser lines, respectively, and emission was acquired with a charged CCD camera . The software VisiView acquisition software  was used to acquire the imaging data. Images were background corrected with an ImageJ-Plugin .Hela cells were transfected with MICU1-YFP constructs (MICU1-WT/MICU1-F/MICU1-K) and MCU-mCherry or EMRE-mCherry. After 2 days, cells were imaged directly using a confocal spinning disk microscope. The MICU1-YFP constructs (MICU1-WT/MICU1-F/MICU1-K) and MCU-mCherry or EMRE-mCherry were excited with 488 and 561 laser lines, respectively. Images were background corrected with an ImageJ-Plugin . Co-localization was measured as Pearson, Manders1 and Manders2 co-localization coefficients using the Coloc2-tool in ImageJ. For Manders 1/2 coefficient, the automated Costes threshold regression was chosen. Manders 1 defines the fraction of YFP-tagged MICU1-WT, MICU1-K or MICU1-F with EMRE- or MCU-mCherry and Manders 2, the fraction of EMRE- or MCU-mCherry overlapping with MICU1-WT, MICU1-F, and MICU1-K.Z-stacks were background corrected with an ImageJ-Plugin  and auto thresholded by using whole stack histogram and segmented using the 3D-Manager of ImageJ. By using the tool ‘3D-ellipsoid fitting' for segmented mitochondria, elongation and flatness factors were determined. The plugin ‘3D Geometrical Measure' was used to measure the volume and surface area of segmented mitochondria. The 3D formfactor (FF) was determined with following equation:Hela cells were transfected with MICU1-YFP constructs (MICU1-WT/MICU1-F/MICU1-K). After 2 days, cells were stained for 10 min with 200 nM MitoTracker Red CMXRos and transfected cell were imaged directly with 488 nm laser (YFP) and a 561 nm laser excitation (MitoTracker Red CMXRos). Whole-cell Z-stacks of the same cells were performed with 561 nm excitation with 200 nm Z-step size. 3D stacks of Micu1-YFP (MICU1-WT/MICU1-F/MICU1-K)-transfected cells were analysed with ImageJ. S stands for the surface area and V for the volume of the segmented mitochondria. Lower values show higher mitochondrial branching and higher values less complexity.where in vitro interaction studies, the Escherichia coli codon optimized genes of human MICU1 97-444 and 97-476 were cloned into pETM11 expression vectors  protease cleavage site). The MICU1 R455K mutant was generated using the QuikChange Site-Directed Mutagenesis Kit. Expression plasmids were transformed into E. coli BL21(DE3) and grown in standard lysogeny broth medium. Protein synthesis was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside at OD600∼0.8. After protein expression for 18 h at 16 °C, cells were collected by centrifugation, re-suspended in lysis buffer consisting of 20 mM Tris (pH 7.0), 300 mM NaCl, 10 mM imidazol, 5 mM β-mercaptoethanol, 10 mM MgCl2 and 0.02% (w/v) NaN3, and lysed by sonication in the presence of lysozyme, Benzonase Nuclease (Sigma-Aldrich) and Protease Inhibitor Mix HP (Serva Electrophoresis). The cell lysate was cleared by centrifugation at 20,000 g for 45 min at 4 °C. The supernatant was loaded onto a HisTrap FF column  attached to an Akta Pure FPLC system  and washed with ten column volumes of wash buffer consisting of 20 mM Tris pH 7.0, 300 mM NaCl, 20 mM imidazol, 5 mM β-mercaptoethanol, 10 mM MgCl2 and 0.02% (w/v) NaN3. The sample was eluted with the same buffer containing 400 mM imidazol. Arginine (50 mM) were added after elution to improve long-term stability of the protein.For the E. Coli Bl21 (DE3) cells harbouring a pET28b-PRMT1 vector containing a amino-terminal His6-tag . This truncated version is identical to human PRMT1 (uniprot ID: Q99873-3), except a single amino acid exchange (H161 is Y in human), and represents the vast majority of expressed sequence tagsD-1-thiogalactopyranoside and incubation overnight at 19 °C. Next day, cell pellets were resuspended in lysis buffer (see above), sonicated and centrifuged for 1 h. The cell lysate was loaded on a pre-packed Ni Sepharose column  attached to an Akta Pure FPLC system  and equilibrated with a 50 mM Tris buffer pH 7.5, 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine and 20 mM imidazole (purification buffer). Nonspecific proteins were removed by washing with ten column volumes of purification buffer. Protein elution was performed by the same buffer containing 500 mM imidazole.Rat PRMT1 with a deletion of the first ten amino acids was produced in 2HPO4/NaH2PO4 pH 8.0, 150 mM NaCl and 2 mM β-mercaptoethanol. For the methylation reaction, recombinant MICU1 (40 μM) and PRMT1 (14 μM) were mixed to final concentrations of 20 and 7 μM, respectively. S-adenosyl-L-methionine  was added to a final concentration of 1 mM. Volumes of the methylation reaction were in the order of several millilitres and depending on the protein yield. The reaction was carried out overnight at room temperature. After methylation, the proteins were buffer exchanged into lysis buffer , 10 mM MgCl2 and 0.02% (w/v) NaN3) using a HiPrep 26/10 desalting column attached to an Äkta pure FPLC system . Arginine (50 mM) was added to stabilize the protein. 2 (w/w)% TEV Protease were added and cleavage was performed overnight at 4 °C. After TEV cleavage, proteins were exchanged into lysis buffer without arginine  NaN3) using a HiPrep 26/10 Desalting column attached to an Äkta pure FPLC system  and then applied onto a Ni-NTA column  and the flow through containing cleaved MICU1 was collected. As the PRMT1 construct does not contain a TEV cleavage site, its His6-tag is not cleaved and PRMT1 can be removed. The flow through was further concentrated to a final volume of 2 ml using 10 kD cutoff Amicon Ultra-15 ml centrifugal filter units (Millipore) in a centrifuge with Swinging-bucket rotor.For PRMT1-dependent arginine methylation, the buffer was exchanged via PD-10 Desalting Columns  to methylation buffer consisting of 50 mM NaA size-exclusion step was performed to remove contaminants using a Superdex 75 16/600 column attached to an Äkta pure FPLC system and equilibrated in MICU1 ITC buffer . After size-exclusion chromatography (SEC), MICU1 was concentrated to a 20 μM solution using a 10 kD cutoff Amicon Ultra-15 ml centrifugal filter unit (Millipore) with centrifugation steps of 5 min at 3,500 r.p.m. in a centrifuge with Swinging-bucket rotor.2 and to a synthetic IML2UCP peptide (residues 147–171 of human UCPs) were determined by ITC on a VP-ITC Microcal calorimeter  at 25 °C with 29 rounds of 10 μl injections (first injection 2 μl). All proteins/peptides were buffer exchanged to MICU1 buffer  before ITC. The MICU1 concentration in the cell was 40 μM for all measurements. CaCl2 and IML2UCP2 concentration in the syringe was 500 μM. For measurement where CaCl2 was titrated to MICU1/IML2UCP2, we used a UCP concentration of 100 μM. The ITC data were analysed with the programme MicroCal Origin software version 7.0 and single site-binding model.Binding affinities of MICU1 to CaClt-test and P<0.05 was considered to be significant.Data shown represent the mean±s.e.m. All experiments were performed at least three times with Ea.hy926 and HeLa, and for experiments with HUVECs and PAECs three different donors were used. For live-cell imaging, numbers indicate the numbers of cells/independent repeats. Statistical analyses were performed by using the unpaired Student's The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Madreiter-Sokolowski, C. T. et al. PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca2+ uptake in immortalized cells. Nat. Commun. 7:12897 doi: 10.1038/ncomms12897 (2016).Supplementary Figures 1-23 and Supplementary Tables 1-3


In support of the hypothesis, we found in the mutated iPSC‐CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC‐CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)‐derived cardiomyocytes (iPSC‐CMs) from SCO2‐mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca To test this hypothesis, we investigated in SCO2‐mutated iPSC‐CMs generated from two babies (who died at 3–4 months) carrying different SCO2 mutations: (i) the ultrastructural changes; (ii) the inotropic responsiveness to β‐adrenergic stimulation, increased [Ca2+]o and AT‐II; and (iii) the Beat Rate Variability (BRV) characteristics. In support of our hypothesis, the diseased cardiomyocytes demonstrated abnormal mitochondrial ultrastructure, functional derangements including arrhythmias, suggesting disturbed intracellular Ca2+ homoeostasis, likely due to ATP deficiency.Derangements in the mitochondrial respiratory chain and specifically in cytochrome c oxidase (COX) are associated with deleterious effects in organs with high energy demands, such as the heart. Consequently, cardiac pathologies are a prevalent outcome of mitochondrial‐related disorders i) an Israeli male baby treated at the Rambam Health Care Campus in Haifa; (ii) an Estonian male baby treated at Tartu University Hospital in Estonia; (iii) a healthy adult Israeli female; (iv) a healthy adult Israeli male. Additionally, hair follicles were collected from a healthy adult Israeli female. iPSC were generated from the donors dermal fibroblasts  or hair keratinocytes (clone N3) previously described Dermal biopsies were obtained from two SCO2‐mutated patients (babies) and two healthy donors (adults): (See details in the Supporting information.2+]i transients and contractions were measured from iPSC‐CMs composing embryoid bodies (EBs), using the IonOptix Calcium and Contractility system , as previously described All methods and protocols are detailed in the Supporting information. In brief, i transient and contraction characteristics than control cardiomyocytes. As seen in Figure 2+]o and AT‐II.The first step in this series of experiments was to determine whether the mutated cardiomyocytes have different o; this intervention augments L‐type Ca2+ current , which in turn increases SR Ca2+ release thereby increasing contractile force n = 12), elevating [Ca2+]o caused positive inotropic and lusitropic effects (P < 0.001), although the [Ca2+]i transients were affected to a lesser extent than contractions  to increased [Ca2+]o.To decipher whether the attenuated response to isoproterenol was due to dysfunctional β‐adrenergic cascade or alternatively—impaired downstream element mediating any positive inotropic intervention, we investigated the effect of elevating i concomitant with contraction cessation, followed by a gradual decline in [Ca2+]i along with resumption (within ~13 sec.) of contractions  had FR, 54% SR and 15% control‐like response. To quantify the response to caffeine, we calculated three parameters: (i) recovery time—the time from the peak of caffeine‐induced [Ca2+]i rise to the first transient; (ii) the per cent change in caffeine‐induced [Ca2+]i signal amplitude, compared to pre‐caffeine amplitude; and (iii) the per cent change in caffeine‐induced Ca2+ signal area, compared to the pre‐caffeine value. In the SR group of both SCO2‐mutated cardiomyocytes, the recovery time was longer (P < 0.05), and the area and amplitude were similar to control cells  which opens RyR2 channels and releases SR Caons Fig. A. In contes Fig. B and C, tes Fig. B and F. tes Fig. C and F; rly Fig. D and E, 2+, implying that the cytoplasm is Ca2+ overloaded, which can thus give rise to delayed afterdepolarizations (DADs) and triggered arrhythmias 2+ overload, transmembrane action potentials were recorded from small clusters of cardiomyocytes in the absence and presence of increasing isoproterenol concentrations. As depicted in Figure 2+ overload concept), in SCO2E140K and SCO2G193S cardiomyocytes  (2/3) isoproterenol concentration, respectively. In addition to DADs, in mutated cardiomyocytes, increasing isoproterenol concentrations led to decreased firing rate (from 10−8 M) and oscillatory pre‐potentials  (4/6). Interestingly, in one of the experiments with SCO2E140K cardiomyocytes, 10−9 M isoproterenol markedly increased the firing rate and eliminated the DADs, but then starting from 10−8 M prominent DADs appeared along with a decline in the firing rate  4/. Interes−9 M–10−6M). Concomitant with the smaller chronotropic response, isoproterenol (10−6M) caused arrhythmias expressed by the irregular spontaneous firing rate  augments the BRV indices versus control cardiomyocytes. To this end, spontaneous electrograms were recorded for 30 min., and BRV analysis performed in the three experimental groups. The first indication of increased BRV in the mutated cardiomyocytes is demonstrated by the dissimilar inter‐beat intervals (IBIs), compared to the fixed intervals in the control cells  values were larger (P < 0.05) in the mutated than in control cardiomyocytes  than in the control cardiomyoctes  of short‐term and long‐term IBI variability, respectively. In support of the higher CV values, the representative Fig. K illustrtes Fig. M–O.2+‐overload resulting in functional derangements and arrhythmias. The major findings in SCO2 cardiomyocytes were as follows: (i) ultrastructural abnormalities, mainly enlarged mitochondria and distorted cristae; (ii) attenuated inotropic response to β‐adrenergic stimulation, [Ca2+]o and AT‐II; (iii) abnormal responses to caffeine administration; and (iv) DADs and triggered arrhythmias.Mutations in the SCO2 gene are among the most common causes of COX deficiency G193S cardiomyocytes demonstrated age‐dependent  abnormalities including enlargement of mitochondria, disorganized mitochondrial cristae, intra‐mitochondrial vacuoles, glycogen deposits and lipid droplets. In contrast, SCO2E140K cardiomyocytes had normal mitochondrial distribution and structure, but large cellular glycogen deposits and lipid droplets. Currently, there is no explanation for these differences; other than that mitochondrial abnormalities seen in mitochondrial diseases are generally heterogeneous and there is often no clear genotype–phenotype correlation. Hence, the mutations in SCO2 are in different domains of the protein; the E140K mutation is close to the copper binding site, while the G193S mutation is in a different segment of the protein, which may also result in different effects of the mutations.Comprehensive TEM analysis in SCO2in vitro recapitulation of the in vivo disease.Mitochondrial structural abnormalities, including massive proliferation, are a secondary effect of the complex IV deficiency, and there is no universal explanation for how these structural changes come about. Possibly, ATP shortage due to COX deficiency in the oxidative phosphorylation pathway leads to a compensatory response causing swelling of mitochondria in an effort to produce more ATP. Further, mitochondrial proliferation is often  seen in mitochondrial diseases, and it is thought to be a compensatory mechanism involving PGC1 alpha/AMPK signalling 2+]o and AT‐II caused prominent positive inotropic and lusitropic effects  activation, augments ICa,L which enhances SR Ca2+ release, thereby augmenting contraction 3, in turn increasing intracellular Ca2+ by opening SR 1,4,5‐IP3‐dependent Ca2+ channels. Collectively, the common denominator of these three positive inotropic interventions is increased SR Ca2+ release.In agreement with our previous studies and others cts Fig.  and 4. I2+ content/release is responsible for the attenuated inotropic responses, we tested the effect (in control and SCO2‐mutated cardiomyocytes) of caffeine which releases Ca2+ from the SR by reducing the activation threshold of RyR2 2+]i along with a transient depression of the contractions, followed by a gradual resumption of contractions. In contrast, the responses of mutated cardiomyocytes were completely different, featuring two major phenomena. Both phenotypes depicted a small and short (compared to control) Ca2+ spike: the first phenotype showed a FR and the second phenotype depicted a SR of contractions. The residual 10–20% (depending on the mutation) exhibited a control‐like phenotype. Thus, while control cardiomyocytes depicted a single typical response to caffeine, the mutated cells exhibited dissimilar (two types) responses to caffeine. These two abnormal responses of the mutated cardiomyocytes were demonstrated previously in sarcomeric‐HCM mutated cardiomyocytes To test the hypothesis that impaired SR Ca2+ handling resulting from the SCO2 mutation and COX impairment. Specifically, the most likely element underlying these Ca2+ handling abnormalities is the ATP‐dependent SERCA pump, responsible for Ca2+ re‐uptake into the SR 2+ stores, leading to attenuated RyR2‐mediated Ca2+‐induced‐Ca2+‐release. Collectively, whereas the three positive inotropic stimuli tested  augment contraction via different pathways  is sufficient to support the excitation‐contraction coupling machinery 2+ levels are insufficient to provide the excessive Ca2+ required to generate a positive inotropic effect. Finally, our findings are consistent with previous studies demonstrating ATP shortage as a cause of HCM 2+ due to impaired SERCA activity. The high Ca2+ concentration activates various downstream proteins and pathways including Ca2+/calmodulin‐dependent protein phosphatase , different nuclear factors of activated T cells (NFAT), Ca2+/calmodulin‐dependent protein kinase (CaMK) and p38 mitogen‐activated protein kinase, which eventually results in HCM and cardiac dysfunction While currently we cannot offer an explanation for the SR and FR phenotypes, it is likely they are both caused by disturbed intracellular Caays Fig. , they shtes Fig. . These r2+ administration) all indicate abnormalities in different cellular pathways converging at the SR. Together with damaged mitochondria demonstrated by TEM analysis, we singled out SERCA, the ATP‐dependent pump responsible for Ca2+ re‐uptake into the SR, as a suspected dysfunctional key element resulting in intracellular Ca2+ overload. In agreement with this conclusion, spontaneous action potential recordings demonstrated DADs, known to originate from Ca2+ overload as well.As depicted in Figure 2+ handling in SCO2‐mutated cardiomyocytes, accounted for by ATP shortage due to the impaired mitochondrial respiratory chain function. These findings are consistent with other HCM iPSC‐CMs studies which demonstrated irregular Ca2+ handling as a fundamental mechanism underlying the disease pathology 2+ abnormalities as a prominent aspect of mitochondrial‐related HCM. Furthermore, the similar properties of different HCM subtypes may indicate a basic common mechanism to which pathological Ca2+ handling can be attributed. Additional research is required to determine whether a common mechanism is indeed responsible for the similar phenotypes or perhaps the resembling outcomes originate from different causes leading to the same HCM disease.In summary, collectively, our novel findings reveal abnormal intracellular CaNone declared.Figure S1 Clinical data of the SCO2 patient at 4 months of age (prior to death).Click here for additional data file.Figure S2 Pluripotency of iPSC derived from the SCO2 patient and a healthy control.Click here for additional data file.Figure S3 Genetic, immunofluorescence and histological characterization of SCO2 iPSC.Click here for additional data file.Figure S4 Genetic and histological characterization of iPSC derived from SCO2 patients and a healthy control.Click here for additional data file.Figure S5 Transmission electron microscopy (TEM) demonstrating ultrastructural abnormalities in SCO2 iPSC‐CM.Click here for additional data file.Figure S6 Basal [Ca2+]i transients and contractions in control and SCO2 iPSC‐CMs.Click here for additional data file.Figure S7 Pluripotency of iPSC derived from human dermal fibroblasts and hair keratinocytes.Click here for additional data file.Figure S8 Effect of isoproterenol on [Ca+2]i transients and contractions in KT and HDF iPSC‐CMs.Click here for additional data file.Table S1. Mean diameter of mitochondria in control and SCO2‐mutated iPSC‐CMs.Click here for additional data file.


In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca Initially relegated to the shadows of cyclic AMP signaling, cyclic GMP has slowly ascended to eminence as an important independent signal in the body, governed by the activities of soluble and membrane guanylate cyclases (MGCs) and phosphodiesterases. Here, we briefly outline the chronological development of the field of mammalian MGCs, focusing on how the modular design of the receptor enzyme adds layers of complexity to its biochemistry and physiological function. Variations in the modules confer flexibility and enable MGCs to command a pervasive signaling role over diverse functions throughout the body. Some of the advancements made by the authors and their collaborators are featured. For more comprehensive coverage, the reader is referred to the authors’ earlier reviews: Sharma , 2010; P; P2010; An observation made five decades ago, that cyclic GMP exists in rat urine  stimulate steroidogenesis, yet do not raise the level of cyclic AMP  activity  of ANF-RGC truly does constitute the hormone’s (ANF) signaling site. A genetic variant of ANF-RGC (GCα), cloned from the adrenal cortex, has a structure that varies in just two amino acid residues from the ExtD of the true ANF-RGC: His338 and Leu364 in ANF-RGC are Glu332 and Val358, respectively. For CNP-RGC, Glu332 in the ExtD is critical for C-type natriuretic peptide (CNP) binding and CNP-dependent catalytic regulatory activity  of the CCD , see below] was shown to be a sensor of atmospheric carbon dioxide  neurocalcin δ (NCδ) was tested as a potential ROS-GC regulator because it shares ~35% sequence identity with GCAPs and is expressed in the inner retina. Ca2+-free bovine brain NCδ has no effect on recombinant ROS-GC1, but the Ca2+-bound form stimulates it with a K1/2 for Ca2+ of 0.8 μM . Because this dimerization domain plays a role in transmitting the signal from GCAP1 to the catalytic module , which bind to an external surface domain. The second signal is internal; a neurocalcin subunit monitors intracellular Can Figure . This CaInsitu hybridization and immunocytochemical studies indicate that GC-D co-expresses with a cyclic GMP stimulated phosphodiesterase, PDE2, that hydrolyzes cyclic GMP as well as cyclic AMP and an ion channel that is gated specifically by cyclic GMP  The Ca2+-responsive system can be reconstituted with recombinant myr-NCδ and ONE-GC.The Ca880-L921 of ONE-GC, a segment encompassing the corresponding V836-L857 binding site in ROS-GC1 (see above), affording direct access to the catalytic domain , GCAP1 stimulates at the upper range of Ca2+  is the most primordial member of the NCS protein family with homologs already present in yeast  stimulates recombinant ROS-GC1, and utterly fails to stimulate ROS-GC2. The binding kinetics between Frq and the M836-C1110 segment of ONE-GC were determined through surface plasmon resonance spectroscopy: KD =0.43 μM; kon (the association rate constant) = 7.14 × 103M−1s−1; koff (dissociation rate constant) = 3.04 × 10−3s−1; and KA (equilibrium association constant) = 2.35 × 106 M−1. Frq therefore may be a soluble modulator rather than a true subunit of the hippocampal MGC. Functionally, a mixed distribution of Frq bound to a minor fraction of the hippocampal MGC and free Frq in the cytoplasm of resting neurons could serve to effectively raise the Ca2+ requirement, extend the Ca2+ range and lengthen the duration of the rise in Ca2+ necessary for stimulation of cyclic GMP synthesis. Modulation of an MGC by myr-Frq suggests that during evolution, Ca2+-sensors that stimulate MGCs at high Ca2+ probably arose before the GCAPs that stimulate at low Ca2+.Co-immunoprecipitation experiments demonstrate that Frq and a ONE-GC-like MGC exist as a complex in bovine hippocampal neurons  and S100B the minor (25%) contributor of Ca2+ signaling by ROS-GC1. The contribution of GCAP1 has not been determined but there is evidence for bimodal Ca2+ operation in bovine and human spermatozoa  and confer Ca2+ sensitivity. The relation between Ca2+ and MGC activity is variable. With ANF-RGC, high Ca2+ stimulates activity without ligand binding whereas ROS-GC1 and ONE-GC can act as bimodal Ca2+ switches; activity is maximal as Ca2+ is depleted, it subsides at midrange [Ca2+]i, then rises once again as [Ca2+] reaches its highest levels. The basis for these different modes of action trace to the identity of the Ca2+ sensor involved: GCAPs, S100B, neurocalcin, frequenin. In a startling arrangement, the conduct of GCAP1 depends upon its MGC partner; it stimulates ROS-GCs at low Ca2+ and inhibits them at high Ca2+, but stimulates ONE-GC at high Ca2+. ROS-GC1 is capable of coupling with many different Ca2+ sensors (Figure 2+/neurocalcin and by bicarbonate. With conservation of the CCD, nearly every MGC responds to bicarbonate. The response of ROS-GC to bicarbonate and Ca2+-modulated GCAPs and S100B in combination exceeds the sum of the individual modulations affording a means to amplify the transductions. It remains to be seen whether bicarbonate can also amplify transduction of orthosteric ligand binding. Chemical modulation and regulation by phosphorylation of MGCs are summarized in Table 2+, GC-G has evolved a remarkable mechanism whereby dimerization and catalytic activity are maximal at a particular temperature in order to sense mild cooling. These transductions encroach on territories of GPCRs and TRP channels. In the former case, MGCs may provide for faster signaling and in the latter, case, amplification. The growing appreciation for MGC signaling throughout the body calls for reclassification of their Ca2+ sensors. NCδ and GCAPs can no longer be considered as strictly NCSs, as their expression is more widespread.How does it perform such diverse tasks? A modular structure makes the MGC extremely flexible and multimodal. Unlike the three-component design of its predecessor second messenger systems: adenylate cyclase and IPs Figure , in partThe reasoning for usage of the present nomenclature for the subtypes of MGCs was explained in our previous review Sharma, : “From iAll authors contributed in the organization, design and writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease. FXN resulting in reduced expression of the nuclear-encoded mitochondrial protein FXN [FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations . Cardiomtein FXN –11. Desptein FXN .2+) cycling properties of cardiomyocytes, to identify potential mechanisms underlying the cardiomyopathy observed in FRDA.Human iPSCs –15 have We generated FRDA-iPSC lines from three individuals with different GAA length repeat numbers Table . We alsoFXN GAA expansions were measured for all fibroblasts and iPSC clones . All clones expressed the pluripotency markers OCT4 and TRA-1-60 Fig.. The int-3. All ces Table . As repolls Fig. . The iPSlls Fig. .MYL2 were significantly higher than MYL7 across all cell lines , which partially blocks L-type Ca2+ channels, prevented an increase in RMSSD, thus confirming that Ca2+ is responsible for the abnormal beat rate variability in line with other studies on PSC-derived cardiomyocytes. In contrast, blocking K+ channels with TEA (10−8 M) did not modify the effect of isoprenaline, with an increase in RMSSD and in cFPD (data not shown). Altogether, these data suggest that FRDA- cardiomyocytes display a significant increase in beat rate variability, demonstrating a potential for cardiac dysfunction, compared to the control cardiomyocytes. These data also suggest that impairment in Ca2+ handling is responsible for the observed electrophysiological phenotype. This was confirmed by assessing Ca2+ transients. In the FRDA-cardiomyocytes significantly lower diastolic and systolic Ca2+ levels and reduced transient amplitude signals were observed compared with control cardiomyocytes  revealed electrophysiological anomalies in the different FRDA lines, with consistent effects in all three clones of all FRDA lines. All FRDA lines showed similar basal beat rates  deficiencies, which are reported in FRDA [2+ uptake and ATP production [2+ levels on mitochondrial function, our data suggests that Ca2+ handling defects could also contribute to the perceived OXPHOS deficiencies. Treatment of FRDA- cardiomyocytes with nifedipine improved phenotypes detected by MEA. Whilst it may seem contradictory that partially inhibiting L-type Ca2+ channels improves cardiomyocytes with low Ca2+ levels, it is possible that nifedipine is acting on other Ca2+ channels independently [2+ levels in the cardiomyocytes [2+ handling machinery of FRDA iPSC-derived cardiomyocytes should unravel the mechanisms behind these phenotypes and identify therapeutic targets.Our data reveal electrophysiological anomalies in FRDA iPSC derived - cardiomyocytes, with an increase beat rate variability. As partial inhibition of L-type Caous iron , 24. Yetous iron . The dat in FRDA , can redoduction , 28. It oduction . Given tendently . This mamyocytes . Further2+ levels in post-mortem FRDA hearts, both describing elevated right ventricle Ca2+ levels, and low-to-normal left ventricle levels in the earlier report versus somewhat elevated left ventricle Ca2+ in a recent report [It might be counterintuitive to relate an increased RMSSD in cultured cardiomyocytes to patient phenotypes. Indeed, patients presenting with cardiomyopathy, including FRDA patients, demonstrate reduced heart rate variability compared to healthy individuals . Howevert report , 33. It All experimental work performed in this study was approved by the Human Research Ethics Committees of the University of Melbourne  meeting the requirements of the National Health & Medical Research Council of Australia (NHMRC) and conforming to the Declarations of Helsinki.The biopsy measured approximately 2–3 mm in diameter and was taken via a needle from the inside of the forearm by a qualified clinician. The risks associated with a skin biopsy are small, however the risks of bleeding and infection were minimised through careful technique, the use of antiseptics and sterile instruments. Minimal pain was experienced at the site once the anaesthetic had worn off and none of the individuals reported any complications following this procedure.iPSCs were generated using skin fibroblasts obtained from FRDA  and control subjects over the age of 18 years by an episomal method as described previously . ReprogrMYH7 for modelling cardiomyopathy [2PO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4, pH 7.35 . Dissociated cardiomyocytes were plated as monolayers on laminin- or Matrigel-coated plates or slides in albumin- and ascorbic acid-free medium. To assess differentiation, we performed qPCR and/or immunostaining of the cardiac markers actin alpha cardiac muscle 1 (ACTC1), troponin T type 2 (TNNT2), troponin I type 3 (TNNI3) and NK2 transcription factor related locus 5 (NKX2.5) to assess presence of cardiomyocytes, and atrial and ventricular myosin light chain 2 (MLC2a/ MLC2v) to assess the proportion of atrial and ventricular cardiomyocytes. TNN and ACTC staining were also used to assess cardiomyocyte morphology. Nuclear factor of activated T cells 4 (NFATC4) was used to assess the presence of hypertrophy in the cardiomyocytes. A visual assessment of beating cells further confirmed the cardiac phenotype.The FRDA-iPSC lines FA6, FA8, FA9 and the control human embryonic stem cell (hESC) line H9 (WiCell)  were maimyopathy . hESCs amyopathy , hence cmyopathy . In the myopathy  was alsomyopathy . FollowiFXN (Hs00175940_m1), MLC2a/MYL7 (Hs01085598_g1), MLC2v/MYL2, (Hs00166405_m1), ACTC1 (Hs01109515_m1), TNNT2 (Hs00165960_m1), TNNI3 (Hs00165957_m1), NKX2.5 (Hs00231763_m1), Glyceraldehyde 3-phosphate dehydrogenase , beta-actin  . The relative quantitation was achieved by applying the comparative CT method (ΔΔCT) whereby the mRNA levels were normalized against the level of GAPDH or ACTB and the control group was used as the calibrator.Total RNA was extracted from cells using the RNeasy Mini Kit , converted to cDNA using High Capacity cDNA Reverse Transcriptase Kit . Q-PCRs were carried out using TaqMan Universal master mix and the 7900HT Fast Real-Time PCR system using TaqMan gene expression assay for Cells were fixed with 4% paraformaldehyde (PFA) or ethanol (OCT-4), blocked in 10% fetal calf serum-PBT, and immunostained using the following antibodies: mouse anti-OCT3/4 (Santa Cruz Biotechnology), mouse anti-TRA-1-60 (Millipore), mouse anti-NESTIN (Millipore), rabbit anti-alpha-fetoprotein , mouse anti-smooth muscle actin , mouse anti-ACTC1 (Abcam), rabbit anti-NFATc4 (Santa Cruz), rabbit anti-MYL2 (Proteintech), mouse anti-MYL7 (abcam). Cells were then immunostained with the appropriate conjugated secondary antibodies . Nuclei were counter-stained with Hoechst-33342 (Sigma-Aldrich) or DAPI (Invitrogen). Specificity of the staining was verified by the absence of staining in negative controls consisting of the appropriate negative control immunoglobulin fraction (Dako).FXN gene was determined by PCR using the Expand Long Range dNTPack  as recommended with 20 ng template DNA, 0.4 μM of EXP-Bam-F 5′AAGGAAGTGGTAGAGGGTGTTTCACGAGGA3′ and EXP-Bam-R 5′TTTGGATCCAACTCTGCTGACAACCCATGCTGTCCACA3′ primers and 1x Q solution . PCR products were electrophoresed on a 1% (w/v) agarose, 1x TAE gel alongside standard DNA markers . Size determination was performed using GeneTools software from SynGene, Synoptics (In Vitro Technologies). The positive control (BAC clone RP11-265B8) and non-expanded alleles in the normal range yielded an 810 bp fragment. The hESC H9, BG01V (ATCC) and the human fibroblast feeders WS1 (ATCC) were included as negative controls for FXN expansion.Genomic DNA for GAA expansion analyses was extracted from fibroblasts and iPSCs using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration and purity of the genomic DNA were assessed using a Nanodrop1000 spectrophotometer (Thermo Fisher Scientific). The size of the GAA expansion in intron 1 of the Dipstick assay for FXN (Mitosciences) was performed as per the manufacturer's instructions . This me2+ channel) and isoprenaline (β-adrenergic receptor agonist) (Sigma-Aldrich). Extracellular field potentials were recorded at baseline and 1 minute after addition of drugs. Data were analyzed offline with MC Rack version 4.3.5 software for beating rate, inter-beat (RR) interval and extracellular field potential duration (FPD). RR interval was defined as the time elapsing between two consecutive beats, and FPD was defined as the time interval between the initial deflection of the field potential and the maximal local T wave. To avoid the influence of beat frequency on FPD, FPD measurements were normalized  with the Bazett's correction formula: cFPD = FPD/√ [Electrophysiological measurements of cardiomyocytes seeded onto laminin-coated 60 electrodes MEA plates were performed by MEA  as previously described . Cells wnterval) . Data we2+ was measured by microfluorimetry . Cells were loaded with the Ca2+ fluorescent dye FURA-2/AM . They were placed in a chamber mounted on the stage of a Motec AE31 inverted fluorescence microscope immersed in medium , at room temperature and stimulated to contract at 1Hz. Excitation light at 340 nm and 380 nm was provided by a 75-watt xenon lamp and filter wheel. Emitted fluorescence (510 nm) was recorded by a photomultiplier tube, with the output current converted to voltage and digitized for subsequent analysis. Background correction was undertaken at the completion of each cell recording and incorporated into analysis protocol. The following Ca2+ parameters were measured: diastolic Ca2+, systolic Ca2+, amplitude of the Ca2+ transient and time constant of decay of the Ca2+ transient (tau).Intracellular Cawww.graphpad.com). Statistical methods utilized were one-way ANOVA followed Tukey's or Bonferroni's multiple comparisons test and t-test. Statistical significance was established as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Data are expressed as mean ± standard error of the mean (SEM). All statistical analyses and graphical data were generated using Graphpad Prism software (v5.04,


There has been a long-standing debate about the extent to which the second messenger inositol 1,4,5-trisphosphate (InsP3) and type 3 InsP3 receptor (InsP3R3) are involved in the opening of Trpc2 channels and in sensory activation of the VNO. To address this question, we investigated VNO function of mice carrying a knockout mutation in the Itpr3 locus causing a loss of InsP3R3. We established a new method to monitor Ca2+ in the endoplasmic reticulum of vomeronasal sensory neurons (VSNs) by employing the GFP-aequorin protein sensor erGAP2. We also performed simultaneous InsP3 photorelease and Ca2+ monitoring experiments, and analysed Ca2+ dynamics, sensory currents, and action potential or field potential responses in InsP3R3-deficient VSNs. Disruption of Itpr3 abolished or minimized the Ca2+ transients evoked by photoactivated InsP3, but there was virtually no effect on sensory activation of VSNs. Therefore, InsP3R3 is dispensable for primary chemoelectrical transduction in mouse VNO. We conclude that InsP3R3 is not required for gating of Trpc2 in VSNs.Signal transduction in sensory neurons of the mammalian vomeronasal organ (VNO) involves the opening of the canonical transient receptor potential channel Trpc2, a Ca Trpc2 is a central transduction element in sensory neurons of the mouse vomeronasal organ (VNO)5 which play important roles in the detection of socially-relevant molecular cues such as pheromones and kairomones8. The recent finding that Trpc2 is also expressed in sensory neurons of the main olfactory epithelium (MOE)10 where it is required in type B cells for the detection of low environmental oxygen11 has sparked renewed interest in its function. Despite two decades of research, the second messenger signaling mechanisms underlying activation of Trpc2 and its corresponding sensory responses are still debated and there is presently no single agreed-upon mechanism for its activation15. Moreover, the behavioral phenotypes of Trpc2 mutant mice are more complex than previously thought16. A complete understanding of Trpc2 signaling mechanisms will be required to fully appreciate the role of Trpc2 in chemical communication and social behaviors.The mammalian olfactory system has evolved two major signaling systems for chemoelectrical transduction: one that depends on cyclic nucleotide-gated (CNG) channel activation and involving cAMP or cGMP signaling, respectively, and another that depends on transient receptor potential (TRP) channel activation, mainly involving the Trpc2 cation channel3) and the type 3 InsP3 receptor  on chemodetection and transduction of mouse vomeronasal sensory neurons (VSNs), and thus on the activation of Trpc2 channels. VSN chemodetection depends on several large G-protein-coupled receptor families (GPCRs), predominantly the vomeronasal type 1 and type 2 receptors, whose activation stimulates phospholipase C (PLC) leading to the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) into InsP3 and diacylglycerol (DAG)18. Trpc2 was initially proposed to function as a Ca2+ store-activated, capacitative Ca2+ entry (CRAC) channel21 and several studies suggested a critical role for InsP3 in VSN chemotransduction27. More recently, a transduction model has been presented in which InsP3R3 is physically linked to Trpc2 and thereby would directly contribute to its activation29. However, because Trpc2 channels are highly localized in VSN microvilli, at a considerable distance from Ca2+ stores, the validity of such a model has been questioned31. Our own studies concluded that Trpc2 is a DAG-activated, Ca2+-permeable cation channel that can be inhibited by Ca2+-calmodulin (Ca2+-CaM) and does neither require InsP3 nor Ca2+ stores for its activation in VSNs32. Since then, there has been considerable evidence for the existence of additional, sequential or parallel VSN signaling mechanisms some of which require elevated intracellular Ca2+ levels and could thus depend on InsP3R3 activation and store-dependent Ca2+ mobilization16. These mechanisms include the activity of Ca2+-activated chloride channels37, several types of Ca2+-activated potassium channels38, as well as Ca2+-activated cation channels39.Here we assess the impact of the second messenger inositol 1,4,5-trisphosphate (InsPItpr3 locus and thus lacks InsP3R341. We used a recently developed method to monitor Ca2+ in the endoplasmic reticulum (ER) by employing a novel, genetically-encoded Ca2+ sensor protein specifically targeted to the ER (erGAP2)43, and we performed simultaneous InsP3 photorelease and Ca2+ monitoring experiments. By applying a combination of state-of-the-art Ca2+ imaging and electrophysiological methods using wild-type and InsP3R3-deficient VSNs, we asked the following: (1) Are intracellular Ca2+ stores significantly mobilized during VSN sensory activation? (2) Does InsP3 induce Ca2+ signals in VSNs? (3) Is InsP3R3 necessary for stimulus-evoked Ca2+ signaling, the generation of sensory currents, and action potential or field potential responses in VSNs? With this approach, we provide compelling evidence that sensory activation of the mouse VNO is largely independent of InsP3R3, ruling out a crucial role for InsP3 signaling in the primary chemotransduction process of mouse VSNs.To resolve these problems and to further define the nature of the signal transduction mechanism in VSNs, we investigated VNO function in a gene-targeted mouse strain that carries a knockout mutation in the 3 receptors (InsP3Rs) and intracellular Ca2+ stores in stimulus-evoked responses of mouse VSNs, we first monitored Ca2+ signals in the ER lumen of isolated VSNs. The ER is the main Ca2+-storage organelle of the cell, as the Ca2+ concentration in ER lumen ([Ca2+]ER) is commonly 104-fold higher than cytosolic Ca2+ ([Ca2+]C). Conventional Ca2+ indicators pose serious limitations for monitoring [Ca2+]ER in terms of Ca2+ affinity, organelle selectivity and/or dynamic range. To overcome these difficulties, we used a novel low-Ca2+ affinity sensor, erGAP2, that can be targeted specifically to the ER lumen42. erGAP2 is a fluorescent, genetically-encoded Ca2+ indicator based on the fusion of two jellyfish proteins, GFP and aequorin. erGAP2 has been optimized for measurements in high Ca2+ concentration environments (Kd for Ca2+  = 407 µM) due to two substitutions on the EF hands of the Ca2+-binding domain , a potent activator of the ubiquitous ryanodine receptors50, to induce Ca2+ release from the ER. We measured F470/F405 ratios in 368 cells that expressed detectable erGAP2 fluorescence. All cells showed a decrease of the F470/F405 ratio in response to a 60-s caffeine stimulus, indicative of a sharp decrease of [Ca2+]ER. In contrast, stimulation with the mix of sulfated steroids (E mix) or HMW did not induce major changes in F470/F405 ratio. Imaging of a representative cell and its F470/F405 ratio trace as a function of time is displayed in Fig. 470/F405 ratio  whereas caffeine produced a robust, 15-fold larger decrease in F470/F405 ratio .We expressed erGAP2 in freshly dissociated VSNs from C57BL/6  mice using a herpes simplex virus type 1 (HSV-1) amplicon vector44 Fig. . A 24-h 44 Fig. . We perfals Fig. . We comp2+ release from the ER, we performed simultaneous measurements of [Ca2+]ER and [Ca2+]C using combined imaging of erGAP2 and the cytosolic Ca2+ dye fura-2. For this approach, we imaged erGAP2-expressing cells loaded with fura-2 and performed triple illumination with 340 nm, 380 nm and 470 nm excitation light and single channel emission at 510 nm , and [Ca2+]ER was estimated by using single F470 dynamics because of the partial F405 overlap with fura-2. We imaged a total of 599 erGAP2-expressing cells and applied 60-s stimuli of either caffeine, HMW, or the sulfated steroid estradiol-3,17-disulfate . We observed robust [Ca2+]C increases, measured as a rise in R340/380, in 15 cells responding to HMW and in 8 cells responding to E1050. These cells also showed [Ca2+]C increases in response to caffeine that were accompanied by simultaneous reductions in F470, indicative of massive Ca2+ release from the ER  than HMW  and E1050 . E1050 even induced a small F470 increase of 0.07 ± 0.3305 a.u., not significantly different from HMW . Importantly, average caffeine-induced [Ca2+]C peaks were similar to those induced by HMW  or even smaller than E1050 peaks . Therefore, the main Ca2+ source contributing to HMW- and E1050-induced elevations of [Ca2+]C does not originate from ER stores.By contrast, changes in F2+ is necessary to generate elevations of [Ca2+]C following stimulation with HMW and E1050, we performed fura-2 imaging in freshly dissociated, non-infected VSNs51. We first established that multiple, 60-s stimulations with HMW or E1050 that were separated by 4-min interstimulus intervals are sufficient to produce robust and repeatable increases in [Ca2+]C , a potent inhibitor of sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), in order to deplete intracellular Ca2+ stores. VSNs were incubated with 30 µM CPA for 20 min in an extracellular medium containing Ca2+. We then performed fura-2 imaging during stimulation with HMW or E1050 in the presence of CPA. We observed robust increases of [Ca2+]C in 4/140 cells stimulated with HMW and in 7/250 cells stimulated with E1050  VSNs. We picked 10 GFP+ cells and 2 GFP– cells. We prepared RNA from each cell, generated single-cell cDNA libraries44, and assessed for gene expression by PCR with gene-specific primers. We amplified Omp PCR products in 8/10 GFP+ cells  or Itpr2 (InsP3R2) genes in all samples, except for one of the GFP− control cells that was positive for Itpr2 (not shown). For comparison, we also sampled 18 cells from InsP3R3−/− VNO (which are GFP−), 8 of which were positive for the Omp PCR . These results indicate that InsP3R3 is the predominant isoform expressed in VSNs, whereas InsP3R1 and InsP3R2 are not co-expressed in these cells. InsP3R2 is likely expressed by supporting and other non-sensory cell types. There was no evidence for upregulation of Itpr1 or Itpr2 in InsP3R3−/− VSNs.InsPVNO Fig. . Importaity Fig. . Next, wlls Fig. , indicatles Fig. , demonstPCR Fig. . We did lls Fig. . We foun3 is capable to evoke Ca2+ elevations in VSNs and, if so, whether this signal would require InsP3R3, we performed flash photolysis experiments with caged InsP3 in InsP3R3+/− and InsP3R3−/− VSNs . We used confocal laser-scanning microscopy to monitor changes in cytosolic Ca2+ and to locally deliver ultraviolet light  stimulation which, in turn, caused photoliberation of caged InsP3 and thereby released the active InsP3 molecule. Cells exhibiting Ca2+ transients in response to either E1050 or HMW were identified as functional VSNs and the regions circumscribed by each cell were outlined and stimulated with UV light  of E1050-activated cells and 15/48 (31%) of HMW-activated cells showed Ca2+ transients after UV exposure  that were stimulated successively with a chemostimulus (HMW or E1050), UV light, and a second chemostimulus . Together, these results show that photolysis of InsP3 can induce transient Ca2+ elevations in VSNs heterozygous for the InsP3R3 mutation but that these responses are absent or strongly reduced in InsP3R3-deficient VSNs. Therefore, InsP3R3 is the predominant isoform mediating InsP3-evoked Ca2+ release in these sensory neurons.To determine whether InsPSNs Fig. . Freshlyght Fig. . A typicion Fig.  demonstrure Fig. . By cont050 Fig. , were laase Fig. , indicatght Fig. . Furtherlus Fig. , UV-evok2+ responses in InsP3R3−/− VSNs. We first monitored Ca2+ signals in the ER lumen of InsP3R3−/− VSNs using ratiometric erGAP2 imaging. We analysed [Ca2+]ER in response to caffeine, HMW or E mix in 67 dissociated VSNs expressing erGAP2 via HSV-1 infection. Caffeine induced strong reductions of F470/F405 ratio, consistent with a decrease of [Ca2+]ER , not significantly different from WT VSNs  tended to be slightly lower in InsP3R3−/− vs. WT cells (0.56–0.75), but this difference was not statistically significant , indicating that increases in [Ca2+]C are not different in InsP3R3−/− cells  reductions for caffeine (−3.76 a.u.), but not for E1050 (+0.09 a.u.) and HMW (−0.33 a.u.) stimuli  in InsP3R3−/− and WT cells  of InsP3R3−/− VSNs, consistent with previous reports of WT VSNs38. The properties of urine-evoked sensory currents were analysed in more detail using 5-s urine applications, a holding potential of −70 mV, and a KCl-based intracellular solution   Fig. . Therefo3R3+/− and InsP3R3−/− VSNs , a major histocompatibility complex (MHC) binding peptide58. SYFPEITHI-evoked responses require type 2 vomeronasal receptors and the G protein Gαo59 but persist in Trpc2-deficient mice64. We found that both ligands evoked robust, phasic-tonic field potentials in InsP3R3+/− and InsP3R3−/− VNO that underwent time-dependent desensitization with prolonged stimulation  has been described41. This strain was intercrossed with C57BL/6 mice for at least twelve times before use41. C57BL/6 mice  or InsP3R3+/− littermates were used as reference mice. Some experiments were performed on OMP-GFP mice (heterozygous for the mutation) in which all cells expressing olfactory marker protein (OMP) are genetically labeled and show robust GFP fluorescence54. All experiments were performed on adult, 6–18 weeks old mice (both sexes).All animal protocols complied with the ethical guidelines for the care and use of laboratory animals issued by the German Government and were approved by the Animal Welfare Committee of Saarland University School of Medicine. All methods were carried out in accordance with the relevant guidelines and regulations. Mice were housed in ventilated cages under a 12:12 hour light/dark cycle with food and water available 2+ imaging of freshly dissociated VSNs was performed as described51. VNO epithelium was detached from the cartilage and minced in PBS at 4 °C. The tissue was incubated (20 min at 37 °C) in PBS supplemented with papain (0.22 U/ml) and DNase I , gently extruded in DMEM (Invitrogen) supplemented with 10% FBS, and centrifuged at 100 × g (5 min). Dissociated cells were plated on coverslips previously coated with concanavalin-A type V . Cells were used immediately for fura-2 imaging after loading them with fura-2/AM  for 60 min, or they were infected with HSV-1 encoding erGAP2 virus and incubated at 37 °C in FBS-supplemented DMEM medium for 24 h before imaging44. Coverslips containing VSNs were placed in a laminar-flow chamber (Warner Instruments) and constantly perfused at 22 °C with extracellular solution Hank’s balanced salt solution  supplemented with 10 mM Hepes (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). Cells were alternately epi-illuminated at 405 and 470 nm for erGAP2 imaging, or at 340 and 380 nm for fura-2 imaging, and light emitted above 510 nm was recorded using a C10600-10B Hamamatsu camera installed on an Olympus IX71 microscope. For simultaneous recordings of [Ca2+]ER and [Ca2+]C, erGAP2-expressing cells were incubated for 60 min with fura-2/AM and sequentially excited at 340, 380 and 470 nm. We recorded emitted light at wavelengths >510 nm. The ratio F340/380 was used as an index of [Ca2+]C and F470 as an index of [Ca2+]ER. Images were acquired at 0.25 Hz and analysed using ImageJ (NIH), including background subtraction, region of interest (ROI) detection and signal analyses. ROIs were selected manually and always included the whole cell body. Peak signals were calculated from the temporal profiles of image ratio/fluorescent values. Results are based on recordings from 3–5 mice for each condition and genotype.Ca2+ imaging were prepared fresh daily and diluted in extracellular solution giving the following final concentrations: HMW fraction, 1:300 dilution; sulfated estrogen mix (E mix): E1050 -estratrien-3, 17β-diol disulphate), E1100 -estratrien-3, 17β-diol 3-sulphate), E0893 -estratrien-3, 17α-diol 3-sulphate) and E0588 (17β-dihydroequilin D 3-sodium sulphate), each at 100 µM ; E1050, 100 µM; caffeine, 50 mM; KCl, 100 mM. Sulfated estrogens were initially prepared in dimethyl sulfoxide (DMSO) and further diluted in extracellular solution. To obtain HMW fraction, 0.5 ml of fresh urine was collected from adult C57BL/6 males 47 and size-fractionated by centrifugation  using Microcon 10-kDa molecular mass cutoff ultrafiltration columns (Millipore). The centrifugation retentate was washed with 0.5 ml of PBS three times and resuspended in 0.5 ml of PBS. Ca2+-free solution was prepared by adding 0.5 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) to the extracellular solution. In some experiments, cells were incubated for 20 min at RT in extracellular solution containing 30 µM cyclopiazonic acid (CPA) to deplete intracellular Ca2+ stores before application of HMW or E1050.Chemostimuli for Ca3, UV laser light was optimally focused using 18 µm thick brain tissue sections loaded with Hoechst 33342  and the semi-automated correction tool of the Zen software (Zeiss). Photolysis of caged InsP3 was achieved by directing UV laser light (1.036 mW) on preselected ROIs (spot diameter ~10 µm) using the Zen software (Zeiss) before reverting back to the visible wavelength laser to resume monitoring of fluo-4 fluorescence. Photorelease of caged InsP3 with ROI spot illumination was performed on single cells previously identified to respond to HMW or E1050. In some experiments, we used UV whole-field illumination  to photorelease InsP3 in a larger area containing multiple fluo-4 loaded cells, in order to record also from IP3R3-deficient cells that did not respond to HMW or E1050 but were potentially sensitive to InsP3 . The estimated intracellular InsP3 concentration after photolysis was expected to be in the 0.1–5 μM range56. Ca2+ changes were generally expressed as relative fluorescence changes, i.e. ΔF/F0 . Images were acquired at 0.5 Hz and analysed using ImageJ (NIH). Peak Ca2+ signals evoked by photoreleased InsP3 were calculated from the temporal profiles of ROI values during the first 10–20 s after a UV stimulus.VNO cells were dissociated and plated on coverslips as described above and loaded with 3 μM caged InsP3/PM  mixed with the same volume of Pluronic F127 in DMSO (10%) in Hepes-HBSS buffer. Cells were loaded with caged InsP3/PM for 30 min at room temperature in the dark followed by an additional 30 min incubation of 2.5 μM fluo-4/AM (Invitrogen) and InsP3/PM. Stock solutions were made in DMSO and kept for up to 1 week stored at −20 °C. The final DMSO concentration did not exceed 0.5%. Coverslips containing VSNs were placed in a laminar-flow chamber (Luigs and Neumann) and constantly perfused with extracellular Hepes-buffered solution. We used an upright scanning confocal microscope (Zeiss LSM 880 Indimo) equipped with a standard Argon laser for excitation at wavelength of 488 nm (fluo-4 excitation) and a UV laser (Coherent) emitting 355 nm (InsP3 uncaging). Images were acquired at 0.5 Hz. Emitted fluorescence was collected between 500 and 560 nm. All scanning head settings were kept constant during each experiment. The UV laser light was coupled to the confocal microscope and focused onto the image plane through a 20 × 1.0 NA Plan-Apochromat water immersion objective (Zeiss). The depth of focus was 16 µm which ensured, together with the region of interest (ROI) diameter, illumination of individual cells. Before starting photolysis of caged InsP44. This virus-based amplicon delivery system was initially developed to overexpress vomeronasal receptors of the V1r, V2r, and Fpr families in VSNs44. To produce viral vectors, a HindIII/EcoRI fragment containing the erGAP2 cDNA was cloned into the herpes simplex virus plasmid pHSVpUC. Packaging and titration of virus particles were performed as reported earlier75. VSN cultures were infected with a multiplicity of infection (moi) ranging between 0.01 and 0.1 one day before use.Virus production, packaging of herpes simplex virus type 1 (HSV-1) vectors and VSN infection was performed as described2 prior to decapitation and VNOs were removed, fixed for 3 h in 4% paraformaldehyde, equilibrated overnight in PBS containing 30% sucrose, embedded in OCT (Tissue-Tek), and snap-frozen in a dry ice/2-methylbutane bath. Frozen tissue sections (16 µm) were collected on glass slides  and stored at −80 °C until use. Sections were post-fixed 15 min in 4% paraformaldehyde, washed 3 times in PBS (10 min each), incubated in blocking solution  for 3 h, and incubated overnight at 4 °C in blocking solution containing anti-InsP3R3 primary antibody . The tissue was then washed 3 times in PBS (10 min each), incubated in AlexaFluor 488 goat anti-rabbit secondary antibody  1 h at room temperature, and in Hoechst  5 min at RT. Sections were mounted using Vectashield Mounting Medium (Vector). Fluorescent images were acquired on a Nikon 80i microscope.Mice were deeply anesthetized with CO44. VSNs were collected using a glass capillary (~10 m tip size) in 1 µl of extracellular solution. Single cells were then transferred to a PCR tube containing 1 µl of diethylpyrocarbonate (DEPC)-treated water. As control, 1 µl of extracellular solution containing no cells was collected with the glass capillary. Samples were immediately frozen on dry ice and kept at −80 °C until use. Second rounds of PCR were performed using the following primers: Omp: GCACAGTTAGCAGGTTCAGCT and GGTTTGCAGTCCTGGCAGC; Itpr1: CGAGGCTGGAAATGAAGGGT and CCACTGAGGGCTGAAACTCC; Itpr2: GGCTGCAAAGAGGTGAATGC and GACGCGATGTCATTTCCGTG; Itpr3: TGCCATGTCCCTGGTGAGC and GACCTGAAGGAAGGGCAGTG. PCR products were sequenced to exclude unspecific amplification or false positives. Total VNO mRNA was obtained from pooled VNO tissue of C57BL/6 adult mice (both genders) using PureLink RNA Mini Kit (Ambion) according to the manufacturer’s instructions. Traces of genomic DNA were digested by incubation with 30 U of DNase I (Fermentas).Single-cell cDNA libraries were prepared as described57. The recording chamber was perfused at a rate of ~1 ml/min with bicarbonate-buffered oxygenated (95% O2/5% CO2) extracellular solution (ACSF) containing (in mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 10 glucose; pH, 7.3; 300 mOsm. The intracellular solution contained, in mM: KCl 140, EGTA 1, Hepes 10, Mg-ATP 2, Na-GTP 1, pH, 7.1; 290 mOsm for urine-evoked responses and: CsCl 140, EGTA 1, Hepes 10, Mg-ATP 2, Na-GTP 1, pH, 7.1; 290 mM for some E1050-evoked currents. Recordings were performed at room temperature using an EPC-9 patch clamp amplifier  and Pulse 8.80 software. In voltage-clamp experiments the membrane potential was clamped to −70 mV, if not otherwise noted. For urine-evoked responses, we chose VSNs from both apical and basal layers. For E1050-evoked responses we focused on VSNs located in the apical layer. In some experiments, we elicited voltage ramps  at the peak of sensory currents. Urine samples were freshly collected from mature C57/BL6 mice (either sex) and frozen for up to three months at −80 °C. Chemostimuli were delivered for 1 or 5 s via multi-barrel pipettes using a pressurized perfusion system . Extracellular loose-patch recordings were performed as described58. Local field potentials from intact VNO (fluid phase) were recorded as described previously63.Whole-cell voltage-clamp or loose-patch recordings from optically identified VSNs were performed in acute VNO tissue slicest test and Mann-Whitney U test were used for measuring the significance of difference between two independent distributions, Wilcoxon signed-rank test was used when comparing two related samples, and Kruskal-Wallis test for three or more independent samples. Analyses were performed using Origin8.6 (OriginLab) or Igor Pro (WaveMetrics) software. Unless otherwise stated, results are presented as means ± SEM. Box-whisker plots show median values, mininimum-maximum outliers, and interquartile (25–75%) ranges.Student’s The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figure S1