These results suggest that other regions of the sodium channel ( Fache et al., 2004 and Lee and Goldin, 2009) and/or interactions mediated by their β subunits ( McEwen and Isom, 2004 and Ratcliffe et al., 2001) may contribute to nodal localization. We have also found that NF186 is remarkably stable mTOR inhibitor at mature nodes, with a half-life of ∼3 months in vitro based on shRNA knockdown (Figure 5B). This is consistent with a recent study demonstrating that NF186, genetically inactivated

in adult mice, exhibits slow turnover, i.e., weeks to months, at CNS nodes of Ranvier (Zonta et al., 2011). These results, and that of a conditional knockout of Trichostatin A chemical structure NF155 (Pillai et al., 2009), the glial component of the paranodes, indicate that mature nodes and paranodes are relatively stable structures. The stability of these various myelinated domains likely facilitates their ability to function cooperatively to ensure the fidelity of saltatory conduction along myelinated axons. The slow turnover of NF186 at nodes contrasts with its rapid turnover in neuron-only cultures, with a measured half-life of less than 1 week based on surface biotinylation studies (Figures S4A and S4B) and shRNA analysis (data not shown). The turnover of NF186 on axons is further accelerated when neurons are cocultured with Schwann cells (Dzhashiashvili

et al., 2007) and in the absence of ankyrin G interactions (Dzhashiashvili et al., 2007); it is also much more rapid at nodes in the absence of ankyrin G interactions based on Bumetanide the turnover of NF/ICAM constructs in transgenic mice (Figure 7A). Interactions with ankyrin G may delay turnover of NF186 by masking internalization signals (Anderson et al., 2005). Interestingly, NF186 turns over much more rapidly at the AIS, with correspondingly larger effects on the integrity of the sodium channel complex, than at nodes (Zonta et al., 2011).

The presence of the flanking paranodes may contribute to the relative stability of NF186 expression at the node, as suggested by its slower turnover at nodes versus heminodes (Figures 5B and 5C). Finally, loss of NF186 is also associated with a corresponding loss of sodium channels (Figure 5C; see also Zonta et al., 2011), indicating that NF186 has a key role in both node assembly and maintenance. This latter role may contribute to the loss of nodal integrity and neurological disability in specific demyelinating disorders (Lonigro and Devaux, 2009). We also show that NF186 is targeted differently to nascent and mature nodes. Whereas NF186 is targeted to newly formed heminodes and nodes by its extracellular segment, it is targeted to mature nodes by its cytoplasmic domain (Figures 6C, 6D, and 7A).