Region-specific proteolysis differentially modulates type 2 and type 3 inositol 1,4,5-trisphosphate receptor activity in models of acute pancreatitis

Abstract

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IPR) by a diverse array of regulatory inputs results in intracellular Ca signals with distinct characteristics. These events allow the activation of specific downstream effectors. We reported previously that region-specific proteolysis represents a novel regulatory event for type 1 IPR (R1). Specifically, caspase-fragmented R1 display a marked increase in single-channel open probability. More importantly, the distinct characteristics of the Ca signals elicited via fragmented R1 can activate alternate downstream effectors. In this report, we expand these studies to investigate whether all IPR subtypes are regulated by proteolysis. We now show that type 2 and type 3 IPR (R2 and R3, respectively) are proteolytically cleaved in rodent models of acute pancreatitis. Surprisingly, fragmented IPR retained tetrameric architecture, remained embedded in endoplasmic reticulum membranes and were not functionally disabled. Proteolysis was associated with a marked attenuation of the frequency of Ca signals in pancreatic lobules. Consistent with these data, expression of DNAs encoding complementary R2 and R3 peptides mimicking fragmented receptors at particular sites, resulted in a significant decrease in the frequency of agonist-stimulated Ca oscillations. Further, proteolysis of R2 resulted in a marked decrease in single-channel open probability. Taken together, proteolytic fragmentation modulates R2 and R3 activity in a region-specific manner, and this event may contribute to the altered Ca signals in pancreatic acinar cells during acute pancreatitis.

Development of a functional salivary gland tissue chip with potential for high-content drug screening

“…Changes in [Ca 2+ ] i were determined by calculating ratios of 340/380 nm (Fig. 4e–j ), as previously described 65 , 66 . An increase in [Ca 2+ ] i after stimulation with either CCh (Fig.…”

“…For individual calcium measurements, SGm were manually removed from MB-hydrogel on days 7 and 14. Calcium imaging on individual clusters was performed as previously described 65 , 66 . Briefly, fresh AIDUCs (day 0) and SGm removed from MB-hydrogel (day 7 and day 14) were incubated with the calcium indicator 5 μM Fura-2 AM for 30 min in culture media at 37 °C.…”

Differential regulation of ion channels function by proteolysis

“…Remarkably, inhibition of proteasome activity failed to eliminate receptor fragmentation, indicating that other proteases, likely inappropriately and prematurely activated digestive enzymes, account for the proteolytic cleavage in acinar cells. Consistent with this idea, using an in vitro acute pancreatitis model where isolated pancreatic acinar cells were incubated with bile salts, R2/3 fragmentation was also readily evident [65]. Notably, in this case, fragmentation was ameliorated by pre-incubation with cell permeable trypsin inhibitors.…”

“…The native structure of the proteolytically cleaved R2 and R3 was also investigated. Given the same overall topology is shared among all three isoforms of IP 3 R, it was not unexpected that R2 and R3, like R1 were shown to retain tetrameric architecture and remain membrane associated after proteolysis [65]. …”

“…In support of this contention, various strategies aimed at inhibiting the Ca 2+ overload, including inhibition of IP 3 R, show protective effects [85–88]. Of note, region-specific receptor fragmentation occurs at an early stage during acute pancreatitis and substantially decreases the total Ca 2+ release mediated by IP 3 R, which consequently reduces the possibility of ER Ca 2+ depletion and the subsequent Ca 2+ influx [65]. These data suggest that proteolytic fragmentation of R2 and R3 may have a protective effect by preventing pathological Ca 2+ signals and consequently delaying the progress of acute pancreatitis.…”