Stimulation of L-type Ca2+ channel in growth cones activates two independent signaling pathways

Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca2+]i than that in adult synaptosomes. Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca2+-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of alpha-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca2+ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca2+-dependent degradation of brain spectrin and the release of alpha-actinin by calpain.