Fig. 8. Model of NCS-1 in Ca²⁺-dependent neurite outgrowth pattern in L. stagnalis. (A) Neurite outgrowth processes, elongation and branching, are dependent on [Ca²⁺]. At basal Ca²⁺ (100 nM), both processes are negligible (Stage 1). Optimal electrical or chemical stimulation increases [Ca²⁺]_i (200-300 nM) and causes both process to be activated (Stage 2). This level of Ca²⁺ would be sufficient to allow binding to the high-affinity sites in NCS-1, thus allowing further Ca²⁺ increases (400-500 nM) through a positive-feedback mechanism such as NCS-1 stimulation of TRPC5 (Stage 3). At this stage, neurite extension is inhibited without affecting branching; but as the morphological determination of branching cannot be observed without extension, the branching also appears stalled. If the levels of Ca²⁺ continue to elevate (micromolar quantities), the likelihood of binding to the low-affinity site in NCS-1 is increased, thereby facilitating voltage-dependent Ca²⁺ channels and consequently raising the levels higher (Stage 4). At this stage, neurite outgrowth processes are both limited. (B) Calibration curve of Fura-2 signal against free [Ca²⁺]_i. From the Ca²⁺-imaging data (Figs 4 and 6), a calibration curve was constructed as described previously (Feng et al., 2002; Grynkiewicz et al., 1985). The arrows indicate the Ca²⁺ levels in PeA cells at either resting (basal) or electrically stimulated conditions with different treatments, and their corresponding stages as described in the model. (C,D) Structure of NCS-1 protein. (C) Tertiary structure of human NCS-1 (also known as FREQ - HUGO) protein (1G8I). Indicated are the N- and C-termini as well as the four EF-hand structures. Ca²⁺ ions are shown as yellow spheres bound to the three functional EF-hands. (D) Schematic structure of the protein showing the four EF-hands is shown below. Of the EF-hands, three are functional (coloured) and one is non-functional (grey). The high-affinity Ca²⁺-binding sites (red), EF3 and EF4, are adjacent to each other and the C-terminus. Binding of Ca²⁺ to EF3 and the low-affinity site EF2 (blue) exposes a hydrophobic crevasse and putative binding pocket for interacting proteins (Bourne et al., 2001).