REGULATION OF EXCITATION-SECRETION COUPLING MECHANISMS

The vesicular release of transmitters and hormones is a fundamental function of all neurons and secretory cells, but the underlying mechanisms are still poorly understood. In excitable cells, elevation of intracellular calcium (Ca) is the trigger for secretion. Depolarization-evoked Ca entry through voltage-gated channels is the best characterized pathway for Ca elevation. In many cells, secretion can also be evoked by stimulating G-protein coupled receptors that release Ca from IP3-sensitive intracellular stores. The amount of Ca contained within stores is very limited and therefore rapidly depleted. Recently, it has been recognized that store depletion generates a signal that activates Ca influx across the plasma membrane. In blood-derived cells, the pathway for store-operated Ca influx has been identified and called CRAC current (for "Ca Release Activated Ca current). Related, but non-identical store-operated currents have been found in other cells. Using patch clamp techniques with simultaneous measurements of intracellular Ca, we have identified a putative store-operated current activated by thapsigargin in catecholamine-secreting chromaffin cells. Ca entry through this pathway can trigger exocytosis in the absence of depolarization. Additionally, the current appears to have a strong facilitatory effect on conventional depolarization-evoked exocytosis. Our hypothesis is that stimulation of G -protein coupled receptors potently regulates secretion in excitable cells via a novel pathway: activation of a store-operated current. The specific aims will test the hypotheses: 1) that the thapsigargin-evoked current is a store-operated current; 2) that physiological agonists can activate a store-operated current; 3) that physiological agonists can trigger or modulate exocytosis via activation of a store-operated current. Determining the role of G-protein coupled receptors in exocytosis will provide important fundamental understanding of secretory processes, including neuronal synaptic transmission. These insights can lead to development of novel strategies for modulation of secretion in normal and pathological conditions.