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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Unknown (R23)
Project #
5R23NS021597-02
Application #
3449776
Study Section
Physiology Study Section (PHY)
Project Start
1984-12-01
Project End
1988-08-31
Budget Start
1985-12-01
Budget End
1987-08-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520