OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage
Volodymyr Tsvilovskyy, … , Shmuel Muallem, Marc Freichel
Volodymyr Tsvilovskyy, … , Shmuel Muallem, Marc Freichel
Published April 1, 2024
Citation Information: J Clin Invest. 2024;134(7):e169428. https://doi.org/10.1172/JCI169428.
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Research Article Cell biology

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

Abstract

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.

Authors

Volodymyr Tsvilovskyy, Roger Ottenheijm, Ulrich Kriebs, Aline Schütz, Kalliope Nina Diakopoulos, Archana Jha, Wolfgang Bildl, Angela Wirth, Julia Böck, Dawid Jaślan, Irene Ferro, Francisco J. Taberner, Olga Kalinina, Staffan Hildebrand, Ulrich Wissenbach, Petra Weissgerber, Dominik Vogt, Carola Eberhagen, Stefanie Mannebach, Michael Berlin, Vladimir Kuryshev, Dagmar Schumacher, Koenraad Philippaert, Juan E. Camacho-Londoño, Ilka Mathar, Christoph Dieterich, Norbert Klugbauer, Martin Biel, Christian Wahl-Schott, Peter Lipp, Veit Flockerzi, Hans Zischka, Hana Algül, Stefan G. Lechner, Marina Lesina, Christian Grimm, Bernd Fakler, Uwe Schulte, Shmuel Muallem, Marc Freichel

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Figure 4

Enhanced cholecystokinin-evoked exocytosis is mediated by Ca2+ release events from TPC2-containing vesicles.

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(A) Model indicating localization of OCaR1 to secretory granules and lysosomes in acinar cells. NAADP-mediated Ca2+ release via TPC2 channels (blue) is monitored by the activity of adjacent Ca2+-activated chloride channels (brown). (B) CCK-8–induced (2 pM) Ca2+-activated Cl– currents in C57BL/6N and OCaR1–/– acinar cells in the absence of extracellular Ca2+. (C) AUC of inward currents over 20 minutes and average amplitude (n = 16 cells, 5 mice per genotype). (D) Model of C-terminal fusion construct of TPC2 and GCaMP6m. (E) Representative (17 images, 3 mice) confocal microscopy images of TPC2-GCaMP6m–expressing acinar cells costained with anti-GFP and anti-Rab27B and merged image. Scale bars: 5 μm. (F) CCK-8 (2 pM)–induced oscillations in GCaMP6m fluorescence in acinar cells from mice expressing TPC2-GCaMP6m. Acute application of Ned-19 (50 μM) abolishes CCK-8–induced responses (n = 99). (G) Representative traces (n = 20, from 100 cells per mouse, 5 mice) from CCK-8–evoked global Ca2+ oscillations in WT acinar cells. (H) Representative traces (n = 20) of CCK-8 (2 pM)–evoked oscillations in GCaMP6m fluorescence in TPC2-GCaMP6m–expressing acinar cells. (I) Frequency and duration of Ca2+ oscillations detected by TPC2-GCaMP6m during stimulation with 2 pM CCK-8 of acinar cells from C57BL/6N (n = 77 cells, 3 mice) and OCaR1–/– mice (n = 135 cells, 4 mice). (J) Current densities elicited by coapplication of PI(3,5)P2 and TPC2-A1-N in vacuolin-enlarged endo-lysosomal vesicles isolated from HEK293 cells overexpressing mTPC2-RFP with or without OCaR1-YFP. Activated currents were blocked with 1 mM ATP (overlapping the basal current) (n = 4–6). Right: Average current densities at –100 mV. Comparison was done by 2-sample t test (C and I) or 1-way ANOVA and Tukey’s post hoc test (J) (*P < 0.05, ***P < 0.001). IM, 10 μM ionomycin.