Regulation of dense core vesicle exocytosis by secretory carrier membrane proteins in neuroendocrine cells

Secretory carrier membrane proteins (SCAMPs) 1-4 are ubiquitously expressed in mammalian cells and a fifth SCAMP (SCAMP5) is expressed primarily in neuronal cells. The E segment connecting the 2nd and 3rd transmembrane spans is the most conserved structural segment and was shown in previous studies to be required for the function of SCAMP2 in regulated exocytosis in mast cells and neuroendocrine PC12 cells. Here, I used PC12 cells as a model system to study the functions of SCAMPs, wherein dense core vesicles (DCVs) undergo exocytosis upon depolarization. Initial findings showed that SCAMP2 localized in the plasma membrane close to docked DCVs and functioned in exocytosis. The work to which I contributed indicated that SCAMP2 could interact with proteins including the small GTPase Arf6 and phospholipase D1(PLD1), which facilitate synthesis of the phosphoinositide PI(4,5)P2 and phosphatidic acid (PA) and are essential for DCV exocytosis. My results showed that the E peptide segment was involved in SCAMP2's function in Arf6 association, PLD activation and late steps of membrane fusion in DCV exocytosis. Following DCV exocytosis in real time by amperometry enabled the demonstration that SCAMP2 regulates the dynamics of nascent fusion pores. In complementary studies, synthetic E peptide was shown to interact electrostatically with PI(4,5)P 2. By analyzing structural variants of E peptide, I was able to correlate binding to PI(4,5)P2 with inhibition of DCV exocytosis by expressing point mutations in full-length SCAMP2. Finally, we applied RNAi-mediated knockdown of SCAMP expression and evaluated how SCAMP deficiency alters exocytosis and in particular to compare how different SCAMP isoforms affect the process when tested alone and in combination. The results indicate an intimate role of both SCAMPs 1 and 2 in fusion pore formation yet also show that other SCAMPS 3 and 5 contribute to regulation as well. Together, the knockdown results imply that SCAMPs are not functionally redundant in DCV exocytosis; individual isoforms may mediate distinct interactions that act in different collaborative combinations in a multi-step process. Overall, my findings support a hypothesis that SCAMPs function ubiquitously in post-Golgi trafficking events by regulating specific membrane domains.