Characterization of store-operated Ca2+ channels in pancreatic duct epithelia

Abstract

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ~ 8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.

Epithelial monolayer culture system for real-time single-cell analyses

“…This result was not expected based on our previous finding that activated SOC are localized in the basolateral membrane (Kim et al. ). We interpreted that overexpression of Orai1 proteins, typically 10–100 times more than endogenous proteins, results in mislocalization of the protein to the apical side.…”

“…This location is consistent with basolateral endogenous SOC puncta visualized with an Orai3‐specific antibody (Kim et al. ). By contrast, in the monolayers with Orai1‐STIM1 overexpression, both apical and basolateral Ca 2+ raised intracellular Ca 2+ levels, implicating that apically localized exogenous Orai1 forms functional SOC at the apical membrane, along with basolateral endogenous SOC proteins (Fig.…”

Ion Channel Signature in Healthy Pancreas and Pancreatic Ductal Adenocarcinoma

“…However, it has been found that SOC-mediated Ca 2+ influx can be a driving force for exocytosis, evoked by trypsin (Kim et al, 2008) in dog PDEC. The same authors have shown the function of SOCs in dog PDEC where the typical inward rectifying current was found, as for other types of epithelial cells (Kim et al, 2013). Furthermore, it was found that STIM1, STIM2, ORAI1, ORAI2, and ORAI3 as well as TRPC1 and TRPV6 are all expressed in dog PDEC, where ORAI3 was shown to be the dominant expressing type (Kim et al, 2013).…”

“…These data suggest that TRPC channels are involved in the SOC entry of pancreatic acini cells and could contribute to fluid secretion. Other TRP channels have been found to be expressed in exocrine pancreas; TRPV6 (Kim et al, 2013), TRPM7 (Yee et al, 2011) and TRPM8 (Yee et al, 2010). However, only the role of TRPM7 is described.…”

Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells

“…4F). SKF 96365 (100 μM), a compound that blocks both SOCs and DAG-activated TRPC channels [66,69] reducing the Ca 2+ influx across the plasma membrane [70], either completely blocked (Fig. 4G, black trace) or strongly decreased the oscillation frequency.…”