Mechanisms Underlying the Neuronal Calcium Sensor-1-evoked Enhancement of Exocytosis in PC12 Cells

Schuichi Koizumi,Patrizia Rosa,Gary B Willars,R.A John Challiss,Elena Taverna,Maura Francolini,Martin D Bootman,Peter Lipp,Kazuhide Inoue,John Roder,Andreas Jeromin

doi:10.1074/jbc.m201132200

Abstract

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. Although NCS-1 is thought to enhance synaptic efficacy or exocytosis mainly by activating ion channel function, the detailed molecular basis for the enhancement is still a matter of debate. Here, mechanisms underlying the NCS-1-evoked enhancement of exocytosis were investigated using PC12 cells overexpressing NCS-1. NCS-1 was found to have a broad distribution in the cells being partially distributed in the cytosol and associated to vesicles and tubular-like structures. Biochemical and immunohistochemical studies indicated that NCS-1 partially colocalized with the light synaptic vesicle marker synaptophysin. When stimulated with UTP or bradykinin, agonists to phospholipase C-linked receptors, NCS-1 enhanced the agonist-mediated elementary and global Ca2+ signaling and increased the levels of downstream signals of phosphatidylinositol 4-kinase. NCS-1 enhanced the UTP-evoked exocytosis but not the depolarization-evoked Ca2+ responses or exocytosis, suggesting that the enhancement by NCS-1 should involve phospholipase C-linked receptor-mediated signals rather than the Ca2+ channels or exocytotic machinery per se. Taken together, NCS-1 enhances phosphoinositide turnover, resulting in enhancement of Ca2+ signaling and exocytosis. This is a novel regulatory mechanism of exocytosis that might involve the activation of phosphatidylinositol 4-kinase.

Highlights

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins
The yeast frequenin, but not vertebrate NCS-1 [14], has been demonstrated to bind to and activate phosphatidylinositol 4-OH kinase, a phosphatidylinositol 4-kinase (PtdIns4K) isoform [1]. This class of enzyme is key to the supply of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and has the potential to regulate phosphoinositide and Ca2ϩ signaling mediated by G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases
The levels of NCS-1 were analyzed in two different PC12 cell clones and in a number of stable transfected clones obtained from N2 cells (Fig. 1A)

Summary

Introduction

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. We demonstrate that NCS-1 enhances UTP- and bradykinin-evoked exocytosis, and this is associated with enhanced agonist-mediated phosphoinositide signaling and potentiated elementary/global Ca2ϩ signaling events.

Results

Conclusion