A role for protein phosphorylation in regulating synaptic transmission and neuro-secretion has been suggested from electrophysiological studies of ion channels and from biochemical studies of protein kinase mechanisms in the synaptic terminal. A model of excitation-secretion coupling includes (1) excitation followed by Ca2+ entry and (2) the coupling of intracellular Ca2+ to release of neurotransmitters and hormones by vesicle exocytosis. The proposed project is designed to study the effects of protein phosphorylation on the coupling between Ca2+ currents and the final secretory event using primary cultures of calf adrenal chromaffin cells. These cells are an ideal model system for studying membrane excitation and Ca2+ dependent secretion. Specific protein kinases to be studied include the cAMP, calcium/calmodulin and calcium/phospholipid-dependent protein kinases. These expriments focus specifically on effects of these kinase systems on: (1) the kinetics of Ca2+ channels and Ca2+ dependent K+ channels (2) the coupling of Ca2+ entry to secretion and (3) the sensitivity of secretion to the internal transient [Ca2+]. Experiments will use new optical and electrophysiological techniques shown to be viable for these studies. The patch-clamp technique permits (1) measurement of single ion channels and whole cell currents from small secretory cells under voltage-clamp conditions and (2) a low-resistance access to the cell interior allowing intracellular dialysis of solutions, ie. purified kinases and drugs. A combination of (a) the patch-clamp technique (b) optical detection with Ca2+ sensitive indicator dyes and (c) light scattering or capacitance measurements, will permit simultaneous measurement of Ca2+ currents, internal [Ca2+] and secretory events under voltage-clamp conditions. Understanding cellular regulation of excitation-secretion coupling is essential for the study of neural and endocrine dysfunctions involving chemical communication between cells and understanding the actions of psychotherapeutically useful drugs. In addition, the calcium/phospholipid kinase is possibly the mediator of the effects of the powerful tumor-promoters, the phorbol esters. Consequently, information may be obtained concerning intracellular receptors of these tumor promoters and their possible mechanisms of action.
