Modulation Of Vesicles Cycle By Calcium And Protein Kinase A In Soma Of Dorsal Root Ganglion Neurons

In neurons and endocrine cells, neurotransmitter/hormone release and synapticvesicle cycle are important processes. The opening of voltage gated Ca²⁺ channel(VGCC) by nerve impulse elicits Ca²⁺ influx, and resulting intracellular Ca²⁺ transienttriggers Ca²⁺ dependent exocytosis. After that, synaptic vesicles undergo endocytosis,recycle, and refill with neurotransmitters for vesicle cycle. Exocytosis and endocytosisare believed to require Ca²⁺, protein kinase and the GTPase dynamin. However,mechanisms of vesicle cycle modulation by interaction of Ca²⁺ and related proteins havenot been determined. Moreover, it remains unknown whether and how evokedexocytosis and endocytosis are regulated in absence of Ca²⁺.Using membrane capacitance measurement, photorelease and measurement of[Ca²⁺]_i and FM dye methods, we studied regulated exocytosis and endocytosis in somaof dorsal root ganglion (DRG) neurons. (1) We found that membrane depolarizationinduces a new form of rapid endocytosis (RE) in the absence of Ca²⁺ and dynamin. TheRE is tightly coupled to preceding calcium-independent but voltage-dependent secretion(CIVDS). The magnitude of RE is proportional to that of CIVDS and stimulationfrequency. Inhibitors of protein kinase A (PKA) suppress RE induced by high frequencydepolarization, while PKA activators enhance RE induced by low frequencydepolarization. Biochemical experiments demonstrate that depolarization directlyup-regulates PKA activity in calcium-free medium. (2) In the absence of extracellularCa²⁺, photoreleased intracellular Ca²⁺ spikes can trigger Ca²⁺ dependent secretion(CDS). However, extracellular Ca²⁺ with physiological concentration inhibits CDS(ECIS) induced by similar Ca²⁺ spikes. The CDS is enhanced by PKA agonist.Moreover, depolarization improves the CDS, which is inhibited by PKA antagonist.Finally, we observed ECIS in rat adrenal chromaffin cells as well.Taken together, our results reveal that there is a Ca2+-and dynamin-independentform of rapid endocytosis, and physiologically extracellular Ca2+ inhibits intracellularCa2+ dependent secretion in DRG neurons. Both forms of vesicle cycle are modulatedby neuronal activity and PKA dependent phosphorylation without Ca2+ influx. Thesefindings extend our understandings about neurotransmitter release and synaptic vesiclecycle.