This application focusses on mechanisms of exocytosis and endocytosis in chromaffin cells. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. Ongoing studies will verify (or disprove) this hypothesis. In all of our experiments to date, catecholamine release is followed by rapid membrane retrieval (tau of seconds). We will investigate whether membrane retrieval rates are similar for large and small levels of secretion, whether membrane retrieval is Ca2+ dependent, and whether known kinases or phosphatases are important in this process. As we are one of the few labs that measuring retrieval rates in real-time, we believe that we have a contribution to make in understanding this important process. Acetylcholine, released from splanchnic nerve terminals within the adrenal medulla, depolarizes chromaffin cells by activating Ca2+- permeable nicotinic ACh receptors, leading to calcium entry. ACh also promotes calcium release from intracellular stores by activating muscarinic receptors. Three different Ca2+-signals summate to trigger release; these are calcium-entry through Ca2+ channels, calcium-entry through Ca2+-permeable nicotinic ACh receptors and calcium-release from intracellular stores following muscarinic ACh receptor activation. In collaboration with Dr. Green, we will investigate the relative importance of each of these signals, as well as how they work together. N-type Ca channels have been found in virtually every neuron. They are known to be involved in neurotransmitter release at a variety of synapses (as well as chromaffin cells), yet detailed biophysical information has been surprisingly difficult to obtain, in part because N-type Ca channels are found in cells with other types of channels. We have available cloned N-type Ca channels stably expressed in HEK cells. In collaboration with Dr. Richard Miller's lab we plan on carrying out extensive studies of N channel gating currents. This grant application may look diffuse but Specific Aims 3 and 4 are collaborative efforts that strengthen the interactions between my lab and those of Drs. Green and Miller and so should be judged in that context.
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