Restriction of 480/270-kD Ankyrin (G) to Axon Proximal Segments Requires Multiple Ankyrin (G)-specific Domains

Ankyrin(G) (−/−) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrin(G) is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrin(G) to axon proximal segments by evaluation of localization of GFP-tagged ankyrin(G) constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin– GFP-tagged ankyrin(G) complexes in nonneuronal cells. A conclusion is that multiple ankyrin(G)-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrin(G) to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrin(G)-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrin(G) polypeptides, contributes to restriction of ankyrin(G) to axon proximal segments as well as limiting lateral diffusion of ankyrin(G)–neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrin(G) also contribute to limiting the lateral mobility of ankyrin(G)–neurofascin complexes. Ankyrin(G) thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrin(B) and ankyrin(G) to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.