Patch clamped single pancreatic zymogen granules: Direct measurements of ion channel activities at the granule membrane
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abstract
BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.
