Regulated exocytosis is the process whereby a cell responds to a physiological signal by releasing a stored product into the extracellular environment through the fusion of secretory granules with the plasma membrane. Exocytosis is fundamental to digestion, salivary secretion, lung maintenance, endocrine release, and synaptic transmission. A specific failure of this system is responsible for the pathology associated with cystic fibrosis. In order to understand and rectify disturbances of exocytosis, the basic mechanism must first be clarified. This proposal uses the well characterized rat pancreas as a model, and is designed to elucidate a coupling between secretory granule electrolyte transport and exocytotic membrane fusion. These secretory granules contain Cl-and K+ channels that are capable of net salt transport when activated. Specifically, the project will determine the mechanism responsible for the activation of the KATP channel in the pancreatic zymogen granule along with the physiological role of these granule ion channels. Using isolated zymogen granules, the KATP channel will be examined electrophysiologically and a G-protein coupled route to activation will be demonstrated. The model to be tested proposes that the granule KATP channel is responsible for controlling net granule salt transport and swelling. Using in vitro assays for membrane fusion, the activation of this granule channel, and net solute transport, will be shown to contribute to the efficiency or rate of the exocytotic event. The experiments will further differentiate between granule transport having a direct contribution to the formation of the initial fusion pore or by simply causing the microscopic pore to proceed to full membrane fusion via the widening and stabilizing of the connection.
