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 3

Acinar amylase hyperexocytosis leading to elevated plasma levels of digestive enzymes is associated with increased spontaneous Ca2+ transients in OCaR1–/– mice.

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Acinar amylase hyperexocytosis leading to elevated plasma levels of dige...
(A) Analysis of spontaneous amylase exocytosis from acinar cells. Acini from C57BL/6N (n = 9, black) and OCaR1–/– (n = 8, red) mice were incubated for the indicated time periods and the fraction of released amylase measured. (B) The total amylase content was unaltered in acinar cells of OCaR1–/– compared with littermate OCaR1+/+ mice (n = 14 preparations per genotype). (C) Plasma cholecystokinin levels are unchanged in OCaR1–/– mice (OCaR1+/+, n = 7; OCaR1–/–, n = 8). (D and E) Representative traces (25 cells per genotype, normalized to the value at 295 seconds) of Fura-2 fluorescence in C57BL/6N (D) and OCaR1–/– (E) acinar cells in Ca2+-free physiological solutions. Carbachol (CCh; 10 μM) was applied at the end of the experiment as a positive control. (F–I) Spontaneous Ca2+ transients in acinar cells of OCaR1–/– mice can be inhibited by 90-minute preincubation with bafilomycin A1 (Baf A1) (C57BL/6N, n = 5; OCaR1–/–, n = 5) (F) or by additional deletion of both TPC1 and TPC2 (OCaR1–/– TPC2–/– TPC1–/– triple knockout) (G–I). (J) Comparison of frequency of spontaneous Ca2+ oscillations between genotypes of the experiments in D–I (C57BL/6N, n = 7; OCaR1–/–, n = 6; C57BL/6N + Baf A1, n = 5; OCaR1–/– + Baf A1, n = 5; OCaR1–/– TPC1–/–, n = 5; OCaR1–/– TPC2–/–, n = 5; OCaR1–/– TPC1–/– TPC2–/–, n = 7). Statistical analysis was done by 2-tailed Student’s t test (A–C) or 1-way ANOVA with Bonferroni’s post hoc test (J) (**P < 0.01, ***P < 0.001).