OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.


Cloning of OCaR1 cDNA
Expressed Sequence Tags (EST) databases were screened for EST clones encoding proteins that showed amino acid sequence homology with proteins of the TRP channel family.EST clones containing cDNA sequences derived from the mouse or human Tmem63a, Tmem63b, and Tmem63c genes were identified.Tmem63a cDNA was cloned (accession number NM_144794), and the encoded protein was named OCaR1 based on its functional role (see Results).Accordingly, structural homologues TMEM63b (accession number NM_198167) and TMEM63c (accession number NM_172583) were named OCaR2 and OCaR3, respectively.

Western blot analysis
Dishes with a confluent layer of transfected HEK293 cells were washed twice with phosphate-buffered saline (PBS) (pH 7.4) were collected from the dishes without trypsinization after adding 150 µl of 2x Laemmli buffer, sheared through a 0.9 mm diameter cannula, and incubated at 37°C for 30 min before being resolved via SDS polyacrylamide gel electrophoresis (SDS-PAGE) (7% separating gel, 4% stacking gel).
Twenty or forty microliters of these samples were loaded.Gels were blotted and proteins probed with anti-GFP primary antibodies (Roche) and detected with horseradish peroxidase-coupled secondary antibodies and the Western Lightning® Chemiluminescence Reagent Plus (Perkin Elmer).Original scans were saved as TIFF files from a LAS 4000 mini (GE Healthcare) and further processed in Corel Draw.

Expression analysis of OCaR1-encoding transcripts using Northern blotting
Poly-(A) + -RNA (10 µg) from the brain and heart of WT and OCaR1 -/-mice were used for Northern blot analysis as described (1).Blots were hybridized with randomly labeled cDNA probes comprising the full length OCaR1 cDNA (full length probes) and exons 15, 16, and 17 of the mOCaR1 cDNA (delta exon 15, 16, and 17 probes).Filters were exposed to x-ray film for 18-24 h.
To analyze expression of OCaR1 in freshly isolated pancreatic acinar cells, we prepared RNA using the RNeasy Mini kit (Qiagen) and performed one-step reverse transcription-PCR (RT-PCR; Invitrogen) using 20 ng of total RNA/reaction.The intronspanning primers UK79 and UK80 (see Supplemental Table 2) were used for amplification of OCaR1 fragments comprising 397 bp.Total RNA isolated from brain (20 ng) was used as a positive control.

Generation of OCaR1 Knock-out and OCaR1-IRES GFP reporter mice
For the construction of the targeting vector (pUK_17), genomic DNA was isolated from R1 ES cells and used as a template for polymerase chain reaction (PCR) amplification of 5′ and 3′ homology arms with Phusion proof reading polymerase (NEB Biolabs).The 5' homology arm was cloned 5' of a loxP site followed by the genomic sequence containing exons 15, 16, and 17 and another loxP site, an IRES-GFP-cassette and an FRT sequence-flanked PGK promotor-driven neomycin resistance gene cassette (neo), and a third loxP site.The herpes simplex virus thymidine kinase cassette (HSVtk) was introduced 5' of the 5' homology arm for negative selection.The targeting construct was sequenced on both strands and no differences were observed compared to the corresponding genomic sequences of the ES cells.Gene targeting in R1 ES cells was performed as described (2).29 of 454 double resistant colonies showed homologous recombination at the OCaR1 locus as confirmed by Southern blot with a 5' probe external to the 5′ homology of the targeting vector.Four of those clones were expanded and genotyped for OCaR1 +/L3F2 using probes for the 3' homology and for the neomycin resistance gene.Injection of the correctly targeted ES cell clone 12E4 into C57BL/6N blastocysts yielded germ line chimeras.Mice carrying the OCaR1 L3F1 allele were obtained following in vivo deletion of the neomycin selection cassette from OCaR1 L3F2/+ mice by mating with FlpeR (3) (shown in Supplemental Figure S4E).This targeted allele is transcribed into a bicistronic messenger RNA.In alternative approach, OCaR1 +/L3F2 mice were mated with Cre deleter (4) mice resulting in effective Cre-mediated excision of exons 15, 16, and 17 (Supplemental Figure S6, C and F) was detected by Southern blotting in OCaR1 +/-and OCaR1 -/-mice.The exons encode the putative transmembrane segment 5, and deletion generates a premature stop codon in exon 18. Specific 3.3-kb OCaR1 transcripts expressed in brain and heart from WT mice (Supplemental Figure S6G) were not detectable in brain and heart from OCaR1 -/-mice confirming effective deletion of the OCaR1 gene.OCaR1 -/-mice on C57BL/6N background (backcrossed for 7 generations) were compared with littermates (indicated as OCaR1 +/+ ) or C57BL/6N mice (Charles River).OCaR1 -/-mice were crossed with mice lacking TRPML1 (see supplemental methods) or TPC1 (5,6) and/or TPC2 (7), respectively, to generate OCaR1 -/-, TRPML1 -/-double KO mice, OCaR1 -/-, TPC1 -/-double KO mice, OCaR1 -/-TPC2 -/-double KO mice and OCaR1 -/- TPC1 -/-TPC2 -/-triple KO mice.Mice were genotyped by PCR.Mice were housed in an animal facility with 12-h light/dark cycle (light on at 7:00 A.M.) and food and water was supplied ad libidum.

Generation of TPC2-GCaMP Knock Add ON mice
Tpc2-GCaMP Knock Add ON mice were generated at inGenious Targeting Laboratory (iTL, New York).To this end GCaMP6 cDNA was inserted in frame within the last exon 25 of the Tpcn2 gene.The expression of Tpcn2-GCaMP6 fusion is terminated by the endogenous stop signal.A FRT-flanked Neo selection cassette was inserted downstream of 3'UTR.Targeted iTL HF4 (129/SvEv x C57BL/6 FLP) hybrid embryonic stem cells were microinjected into CD-1 blastocysts.Resulting chimeras with a high percentage agouti coat color were mated to C57BL/6N WT mice to generate germline Neo-deleted mice (Tpc2-GCaMP +/T ).Tpc2-GCaMP +/T mice were backcrossed with C57BL/6N WT mice for one more generation and then used for AP-MS analysis.

Organellar proteomics
Acinus cells were prepared from freshly isolated mouse pancreas tissue as described (9) (with a modification that the cells after the collagenase digestion were resuspended in Ca 2+ -free solution A supplemented with 0.25 mM EGTA), collected by centrifugation and snap-frozen.600 mg cell pellets were later homogenized in 8 ml 320 mM mannitol / 10 mM Tris HCl / 0,1 mM MgCl2 pH 7.1 (supplemented with protease inhibitors), first using a glass potter (15 strokes, tight pestle) and mild bath sonication, and then a Cell Cracker homogenizer (HGM/EMBL Heidelberg; 10 passages with a 8,006 mm ball).
The homogenate was centrifuged at 1000 x g for 5 min and the supernatant then separated on a 22 ml 0-30% linear Optiprep gradient in homogenization buffer for 1 h at 30,000 rpm (ultracentrifugation in a Beckmann SW 32 Ti rotor).26 fractions of 1 ml were harvested with a peristaltic pump, each diluted in 3.8 ml lysis buffer (10 mM mannitol, 10 mM Tris HCl pH 7.4, 1 mM EGTA/EDTA, supplemented with protease inhibitors) and incubated for 15 min followed by brief bath sonication.Lysates were then subjected to fractional sedimentation by ultracentrifugation at increasing velocity and run time.Visible membrane pellets, as well as TCA-DOC protein precipitates of three fractions above the top of the gradient were collected, dissolved in Laemmli buffer and shortly run on SDS-PAGE gels (42 samples total).After visualization by silver staining, upper and lower halves of each lane were excised and in-gel digested with trypsin.

Laser-capture microdissection
A PALM laser microdissection system with autocatapult and Robocut Software (Zeiss, Oberkochen, Germany) was used for microdissection as described (10).Eight-to nineµm serial sections from mouse pancreas were transferred to PALM membrane slides (Zeiss), fixed with ethanol and stained with cresyl violet.Stained slices were stored in ethanol at 4°C until microdissection (30 minutes to 4 hours).Fifteen minutes before microdissection, individual slides were air-dried at 21°C.Pancreatic acinar cell clusters consisting of 3-10 cells were microdissected and catapulted into the sterile adhesive caps of 0.5 ml tubes (Zeiss).About 40 chippings of ~1,500 to 7,500 µm 2 were collected per preparation.For RNA isolation, chippings were dissolved in Buffer RLT (Qiagen, Hilden, Germany) after microdissection and snap-frozen on dry ice.Total RNA was extracted from individual caps using the Qiagen RNeasy micro kit with deoxyribonuclease (DNase) on-column digestion.Fifty percent of the eluted RNA was used for one-step reverse transcription-PCR (RT-PCR; Thermo Fisher Scientific).The intron-spanning primers UK79 and UK81 were used for amplification of a specific OCaR1 fragment comprising 397 bp.All oligodeoxynucleotide primers used in this study are listed in Suppl.Table 2. Total RNA isolated from brain (20 ng) was used as a positive control, and H2O was used as the negative control.

RT-PCR expression analysis
For RT-PCR analysis, RNA isolation and expression analysis in mouse cardiomyocytes, mouse cardiac fibroblasts, and mouse platelets were performed as already described (11,12).Mouse peritoneal mast cells and bone marrow-derived mast cells were prepared as described previously (13,14).RT-PCR was performed as with RNA samples obtained from laser capture microdissection.

Induction of chronic pancreatitis
For the induction of chronic pancreatitis, mice received for two consecutive days (d1, d2) eight hourly, intraperitoneal (i.p.) injections of 0.1 µg cerulein/g body weight (4030451; Bachem Holding AG, Bubendorf, Switzerland) diluted in phosphate buffered saline according to the scheme in Figure 5C.Blood was taken at 0h, 8h, 24h, 32h, and at sacrifice 96h (d5) after the first injection to quantify damage-induced pancreatic enzyme release.Serum was diluted 1/10 with 0.9% NaCl.Amylase activity (AMYL2 Cobas, Roche in Units/l) was quantified by a colorimetric assay according to the IFCC method.Lipase activity (LIPC Cobas, Roche in Units/l) was quantified by DGGR substrate-based assay.After sacrifice, pancreatic tissue was weighted for relative pancreatic weight determination (pancreas weight/body weight depicted in %), fixed with paraformaldehyde, and embedded in paraffin for histological analyses.2 µm-thick tissue sections were prepared and stained with Hematoxylin & Eosin (H&E) for morphological examination.For detection of fibrosis, deparaffinized and rehydrated slides were counterstained with hematoxylin and incubated for 1 h at room temperature with 0.1% Picro-Sirius red solution (0.1% direct red 80 and 1.3% picric acid in water; Sigma, Munich, Germany).After washing with 0.5% glacial acetic acid in water, slides were dehydrated, cleared in Xylene, and mounted.Pictures (200x magnification, n = 10-15 per mouse) were taken using Axiostar Plus (Carl Zeiss, Göttingen, Germany) and % of Sirius red stained area/picture was quantified with ImageJ.Averages for each mouse were made and used in GraphPad Prism for statistical analysis.Comparisons were made between four non-treated OCaR1 -/-and four C57BL/6N controls.

Induction of acute pancreatitis (AP)
Blood was collected from overnight starved OCaR1 +/+ and OCaR1 -/-mice to analyze basal amylase levels.After a recovery time of 3 days, mice were starved overnight and then administered seven injections (i.p., hourly over 7 hours) of cerulein (50 ng/g body weight) (15).One and 16 hours after the last cerulein injection, blood was collected for the measurement of serum amylase.After the final blood collection, mice were sacrificed and the pancreas was excised in order to determine pancreas dry/wet weight as a measure for edema formation.

Analysis of Ca 2+ transients
Acinar cells were loaded with Fura-2 by incubation with 4 µM Fura-2 AM (Thermo Fisher Scientific, Darmstadt, Germany) for 30 minutes at room temperature and were perfused (room temperature) with physiological solution (PS, i.e.PSA without BSA and soy bean trypsin inhibitor).[Ca 2+ ]i was measured following excitation at 340 and 380 nm, and the emitted light was collected by a digital camera with a cutoff filter at 510 nm and analyzed with the Axiovision Software (Zeiss) and Origin Software (OriginLab).The data is represented as normalized (divided by fluorescence ratio at t = 295 s) example traces and summary statistics.Each datapoint on the summary statistics represents one cell isolation, which is one mouse.Statistical testing was performed on mice.10 µM carbachol (Cch) was applied at the end of the experiment as a positive control for the assay and cell viability, nonresponding cells were excluded from analysis.
Whole lysosomal electrophysiology was performed in isolated endolysosomes using a modified patch-clamp method (17).Transfected COS-7 cells were released and replated on Petri dishes and incubated with culture media for 1 h to allow attachment.
Thereafter, the cells were treated with 1 μM vacuolin-1 (a lipid-soluble polycyclic triazine) that selectively increases the size of endosomes and lysosomes.After

Single channel recordings of mechanosensitive currents
To examine stretch-activated PIEZO1 currents, PIEZO1 was transfected into N2a-PIEZO1-KO cells (gift from Gary R. Lewin) (18) using the calcium phosphate method.
For transfection, growth medium was replaced with transfection medium consisting of DMEM, 10% calf serum (Thermo Fisher), and 4 mM L-glutamine.Each coverslip had 0.6 µg DNA diluted in 100 µl water and mixed with 10 µl 2.5 M CaCl2.Subsequently, 100 µl 2x BBS (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.0) was added and vortexed.The resulting DNA mix was added to the transfection medium.After 3-4 hours at 37ºC (5% CO2), the transfection medium/DNA mix was replaced with regular growth medium.PIEZO1 function was assessed 48 h after transfection.
Stretch-activated currents were recorded as described (19) at a holding potential of -80 mV in the cell-attached patch-clamp configuration (sampled at 20 kHz and filtered at 1 kHz) using an external solution consisting of 140 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES adjusted to a pH of 7.3 with KOH.The recording pipettes were coated with Sylgard, had a resistance of 6-8 MΩ and were filled with a solution containing 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM TEA-Cl, and 10 mM HEPES adjusted to pH 7.3 with NaOH.Pressure stimuli were applied with a 1ml syringe, and the pressure was measured with a custom-made pressure sensor.
Single channel amplitudes were determined as the difference between the peaks of Gaussian fits of the trace histograms of 5 consecutive stimuli using Fitmaster software (HEKA Elektronik GmbH).

Transcriptome analysis
RNA was isolated from the pancreas of three 3-week-old male WT (C57BL6/N) and OCaR1 -/-mice, using TRIZOL reagent (Thermo Fisher Scientific, Germany).The integrity of the RNA samples was verified using a 2100 Bioanalyzer system (Agilent Technologies, USA), and the RNA integrity number (RIN) was between 8.20 and 9.10.
A cDNA library was generated using the NEB Next RNA Master Mix Library Prep Kit for Illumina with the NEB Next Oligodeoxynucleotides for Illumina (New England Biolabs, Germany).First-strand synthesis was conducted employing 2.5 µg RNA that was polyA-RNA selected.The read length of the RNA-seq (Hiseq2000 machine using the standard Illumina sequencing by synthesis chemistry) was single-read, 51-base pair reads, SE.We obtained 30.0-33.9 million reads per sample.All reads were alignesd against the mouse genome sequence (mm10, genome-build GRCm38.p5)using the splice-aware STAR read aligner (version 2.6.1d)(20) with a high-mapping rate (>95%).Among them, 56.6-62.0%were uniquely mapped, and we used them in subsequent analysis.
To assess the differential gene expression, only aligned reads covering exons in positive-strand orientation were counted with featureCounts (version 1.6.3)(21).Differential gene expression analysis was performed with DESeq2 R-package (22).
Genes showing counts less than 100 were removed across all samples.A gene was considered differentially expressed if the FDR adjusted p-value was < 0.05.
MS analysis was carried out on a QExactive MS setup and processed essentially as described for organellar proteomic analysis (in the methods section of the main manuscript and the supplement) with the following modifications: peptide samples were each dissolved in 13 µl 0.5% trifluoroacetic acid each, 5 µl thereof injected; aqueous organic gradient: 6 min 3% B, 60 min from 3% B to 30% B, 10 min from 30% B to 40% B, 5 min from 40% B to 50% B, 5 min from 50% B to 99% B, 5 min 99% B, 5 min from 99% B to 3% B, 10 min 3% B; C18 column: 24.5 cm packed in an equally dimensioned emitter (CoAnn Technology); full MS (precursor spectra): maximum injection time 100 ms; dd-MS2 (fragment spectra): maximum injection time 200 ms, loop count 10 (Top10), minimum AGC target 8,000 (intensity threshold 40,000), dynamic exclusion 30 s. Extracted peak lists were searched against the in-house modified UniProt reference proteome database (UP000000589) + cRAP from The Global Proteome Machine (GPM; http://ftp.thegpm.org/fasta/cRAP/crap.fasta).Labelfree quantification of proteins was carried out as described in (24).

Trpml1
Knock out mice Mice with a Trpml1 null allele were generated by flanking exons 2-5 of the Trpml1 gene with loxP sites using gene targeting in FLP C57BL/6 (BF1) ES cells (at inGenious Targeting Laboratory, iTL) to obtain five heterozygous (Trpml1 +/flox ) ES cell clones, of which one was used to generate heterozygous mice.Correct targeting, deletion of FRT-flanked NEO cassette as well as of exons 2 to 5 was verified by PCR based genotyping startegies.Trpml1 +/flox mice were crossed with Cre deleter mice (8) to obtain Trpml1 +/-and eventually Trpml1 -/-mice.

[
Ca 2+ ]i is given as the 340/380 ratio and was normalized by dividing the values by the initial basal values at time point 295 s.The frequency of spontaneous and agonistevoked (e.g 300 fM cholecystokinin or 100 nM carbachol) Ca 2+ transients were determined by the number of oscillations per cell within 1450 s.The duration and amplitude of these oscillations were analyzed as follows: the exact amplitude and width of each individual Ca 2+ transient was determined and plotted in a frequency histogram with a binning of 0,1 normalized F340/F380 ratio or 20 seconds for amplitude and width, respectively.The averages of these values were calculated for each individual cell preparation.
1 h treatment with vacuolin-1, enlarged lysosomes were released by quickly pressing patch pipettes against the cell and pulling back.Patch pipettes had a resistance of 6-8 MΩ when filled with the pipette solution.The enlarged lysosomes were identified by monitoring RFP and/or mCherry fluorescence.After formation of a giga-ohm seal, capacitance transients were compensated by Axopatch 200B patch clamp amplifier (Molecular Devices, Sunnyvale, USA).For breaking into the lysosomes, quick voltage steps of +350 mV were applied.The whole lysosomal configuration was confirmed by reappearance of the capacitance transient.The current was recorded by 400 ms rapid alterations of membrane potential (RAMP) from −100 to +100 mV from a holding potential of 0 mV at 4 s intervals.The whole-lysosomal currents were filtered at 1 kHz with an internal four-pole Bessel filter, sampled at 5 kHz, and stored directly to a hard drive by Digidata 1322.Currents were measured with an Axopatch 200B patch clamp amplifier that was controlled by pCLAMP9 software (Molecular Devices, Sunnyvale, USA).The methodology of lysosomal current recordings in HEK293 cells is described in the main methods section.

Figure S4 (related to Figure 2 )
Figure S4 (related to Figure 2): OCaR1 expression in acinar cells and localization in lysosomes and secretory granules at the apical pole of acinar cells (A) Analysis of subcellular localization of OCaR1-eYFP fusion proteins in pancreatic acinar cells from offspring derived from zygotes transduced via perivitelline injection with a lentiviral vector (inset) expressing OCaR1-eYFP under control of the pgk promoter.OCaR1-eYFP fluorescence is localized at the apical pole of acinar cells (middle panel).Plasma membrane (PM) is stained using Cell Mask TM dye (right panel).No comparable fluorescence was seen in acinar cells derived from WT mice (not shown).Scale bar 10 μm.(B) Magnification (left panel) of the inset in the merged image (middle panel) indicates membraneous and vesicular localization of OCaR1-eYFP in acinar cells.Scale bar 10 μm.(C) Line-profile of fluorescence intensity values along the dotted line in the merged image shown in B. Representative image from 7 experiments.(D) DIC images (upper panels) and GFP fluorescence (lower panels) in acinar cell clusters of WT mice (left) and mice homozygous for the OCaR1-IRES-GFP allele (OCaR1 L3F1 allele, right).Scale bar 10 µm.(E)In the OCaR1-IRES-GFP allele, which originates after removal of the NEO cassette from the OCaR1 L3F2 allele (see FigureS6B), exons 15, 16 and 17 are flanked by loxP sites (filled triangles), followed by a Splice Acceptor (SA)-internal ribosomal entry site (IRES)-GFP cassette to render GFP as expressional reporter under control of the endogenous OCaR1 promoter.(F) RT-PCR-analysis of OCaR1 expression in microdissected mouse pancreatic acinar cells.Cresyl violet stain of pancreatic cryosections after laser capture microdissection (LCM; scale bar, 25 μm, left).Amplification of OCaR1-specific transcripts (397 bp) from RNA of microdissected acinar cells (3 independent preparations #1, #2, #3) or brain (positive control) using One step RT-PCR (right).

Figure S6 (related to Figure 3 - 5 )
Figure S6 (related to Figure 3-5): Generation of OCaR1-deficient and OCaR1 IRES -GFP reporter mouse lines (A) Intron-exon distribution of OCaR1 (exons shown as vertical lines) on mouse chromosome 1.The OCaR1 gene extends over 32 kb and exhibits 23 exons, of which 22 exons are coding.The start codon ATG is located in exon 2. The transcript length covers 3,306 bp and the encoded OCaR1 protein comprises 804 residues.(B-G) A Cre-loxP based strategy was used to excise exons 15, 16 and 17, thereby inducing a stop codon in exon 18 of the OCaR1 gene.(B) The WT OCaR1 allele, targeting construct, and recombinant OCaR1 L3F2 allele.Translated exons (not in scale) are shown as filled boxes.In the OCaR1 L3F2 allele, exons 15, 16 and 17 are flanked by loxP sites (black triangles); a FRT site (gray triangles)-flanked PGK-neor cassette is located downstream of the third loxP site.A, AflII, B, BamHI; K, KpnI.Probes and sizes of genomic DNA fragments as expected for Southern blot analysis are indicated.HSVtk, herpes simplex virus thymidin kinase.SA-IRES-GFP, splice acceptor-internal ribosomal entry site, followed by the cDNA of the green fluorescent protein.5', 3' and Neo-probes are indicated as black bars.(C) Cre-expression leads to the deletion of exons 15, 16 and 17 and to the conversion of the OCaR1 L3F2 allele to the OCaR1 L1minus allele.(D,E) Identification of the recombinant OCaR1 L3F2 allele in recombinant ES cells (D) and mice (E) by Southern blot analysis using 5' and 3' probes placed externally to the targeted sequence and a neo probe that is directed against the internal PGK-neor cassette.(F) Cre expression in mice resulted in the conversion of the 5.7-kb fragment of the OCaR1 L3F2 allele to a 10.6-kbfragment that is characteristic for the OCaR1 L1minus allele (5' probe; KpnI digest, indicated in C).The 3' probe detects the alteration of the 7.0 kb fragment of the OCaR1 L3F2 allele to a 10.6-kbfragment of the OCaR1 L1minus allele (KpnI digest).In the following, mice carrying the OCaR1 L1minus allele are labeled as OCaR1 +/-and OCaR1 -/-.Cre activity also leads to the excision of the PGK-neor cassette so that the 6.2-kb neo probe signal detectable for the OCaR1 L3F2 allele is absent in OCaR1 +/-and OCaR1 -/-mice (AflII digest).(G) Northern blot analysis of poly(A) RNA isolated from brain and heart from WT (+/+) and OCaR1 -/-(-/-) mice hybridized with three OCaR1-specific probes that cover the full length mRNA (full length probes) and a probe that exhibits the coding sequence of exons 15, 16 and 17 (delta exon 15, 16, 17 probe), respectively.OCaR1 specific 3.3-kb transcripts were identified in WT brain and heart, whereas no signal was detected in brain and heart of OCaR1-/-mice.Gapdh was used as a loading control.

Figure S10 :
Figure S10: The effects of the Orai blocker GSK-7975A on intracellular calcium dynamics in the presence or absence of OCaR1.(A) Representative traces (25 cells per genotype) of Fura-2 fluorescence in pancreatic acinar cells of OCaR1 +/+ mice without and (B) with 10 min preincubation with 10 µM GSK-7975A under basal conditions with 2 mM extracellular Ca 2+ .(C) OCaR1 -/-littermates from the experiments in (A) and (D) with GSK-7975A preincubation.(E) and (F), similar as (A) and (B) but with C57BL/6N animals and (G, H) OCaR1 -/-animals that are not littermates.(I) Peaks per cell from the experiments in (A-D), n = 3 mice per condition.(J) Peaks per cell from the experiments in (E-H), n = 4 mice per condition.Statistical analysis was done by two-tailed Student´s t-test (*p≤0.05;** p<0.01).

Figure S11 :
Figure S11: The effects of CRAC blocker GSK-7975A on intracellular calcium dynamics of C57BL/6N and OCaR1 -/-acinar cells.Representative traces (25 cells per genotype) of Fura-2 fluorescence in pancreatic acinar cells stimulated with 300 fM CCK-8 in 2 mM extracellular calcium from (A) C57BL/6N mice in control conditions and (B) with 10 µM GSK-7975A.(C) and (D) as (A) and (B) but on cells isolated from OCaR1 -/-mice.(E, F, G, H) similar experiments as in (A-D) without extracellular calcium (0.25 mM EGTA) of C57BL/6N mice in (E) control conditions and (F) with 10 µM GSK-7975A.(G) Cells isolated from OCaR1 -/- mice in control conditions and (H) with 10 µM GSK-7975A.(I) The number of calcium peaks per cell (J) the amplitude and (K) the width of the peaks observed in (A-H), n = 3 mice per group.(L) Representative traces (n = 20) from Fura-2 responses on 1 nM CCK-8 in acinar cells from C57BL/6N mice without and (M) with application of 15 µM GSK-7975A during the plateau phase.(N) Representative traces (n = 20) from Fura-2 responses on 1 nM CCK-8 in acinar cells from OCaR1 -/-mice without and (O) with application of 15 µM GSK-7975A during the plateau phase.Note the oscillatory calcium pattern in these OCaR1 -/-cells.(P) Fura-2 fluorescence representing the average response of acinar cells isolated from C57BL/6N mice to 1 nM CCK-8 (in 5 mM extracellular calcium) with and without application of 15 µM GSK-7975A after the initial calcium peak, as in (L and M).(Q) Responses from acinar cells isolated from OCaR1 - /-mice stimulated 1 nM CCK-8 with and without the application of 15 µM GSK-7975A after the initial calcium peak as in (N and O).(R) The average response in the plateau phase at t = 2000 s of the experiments in (P) and (Q), n = 4 mice per group, two sample t-test, ns p>0.05, *p˂0.05,**p˂0,01.

Figure S18 :
Figure S18: Intracellular calcium dynamics during thapsigargin-induced passive depletion of ER in C57BL/6N and OCaR1 -/-acinar cells.(A) The average effect of 10 µM thapsigargin (Tg) on pancreatic acinar cells in calcium-free extracellular conditions.The observed slow calcium transient due to ER depletion seems larger in OCaR1 -/-acinar cells.(B) After 90 min preincubation of the cells with 100 nM Baf A1 to disrupt calcium storage in acidic organelles, the acinar cells of C57BL/6N or OCaR1 - /-mice were exposed to 10 µM thapsigargin to induce passive ER calcium depletion.OCaR1 -/-acinar cells treated with Baf A1 seem to have similar calcium capacity in their ER compared to C57BL/6N cells.(C) The maximum amplitude reached of the experiments in (A) and (B).The traces were baseline corrected by subtracting a linear baseline from t = 295 s (immediately before Tg application) and the lowest point at t = 2700 s immediately before the application of Cch.(D) The area under the curve from the traces in (A) and (B).The total calcium released from the ER of OCaR1 -/-cells is higher than in the C57BL/6N cells.This effect is not present when the cells are treated with Baf A1. (C57BL/6N; Tg: n = 12, Tg + Baf A1: n = 5.OCaR1 -/-; Tg: n = 14, Tg + Baf A1: n = 4).Two-way Anova with Bonferroni corrected pairwise post-hoc comparison, (n.s.p>0.05, **p˂0.01).
GCT CAC CAT CC AB_90 GTC CTC CTT GAA GTC GAT GC UK79 CAG CTT TGG GAG GAC AAC G UK81 GCA GAA CTG GCC ACA AGG