Carbon fiber amperometry in the study of ion channels and secretion

Activation of Ca2+ channels in the plasma membrane or on internal Ca2+ stores raises cytosolic Ca2+ concentration ([Ca2+]c). Among diverse functions of Ca2+ signals, the induction of exocytosis-the process in which the contents of secretory vesicles are released by their fusion to the plasma membrane-is one of the most important. For example, in neurons and endocrine cells, it allows intercellular communication and secretion of biomolecules. Exocytosis can be detected by several physical and chemical means. By chemically oxidizing the released secretory products at a fixed electrode potential, carbon fiber amperometry provides excellent temporal and spatial resolution in detecting exocytosis. This method, together with other biophysical techniques such as patch clamp and Ca2+ microphotometry, has greatly contributed to our understanding of the molecular mechanisms involved in the stimulus-secretion coupling. However, amperometry can be performed only on cells that secrete oxidizable molecules. To overcome this limit, we have developed a protocol of loading cells with exogenous neurotransmitters that readily oxidize on a carbon electrode. Several cell types have been successfully loaded, exocytosis of secretory vesicles has been demonstrated, and in pancreatic duct epithelial cells, the modulatory signals of exocytosis have been studied in detail.