The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

Nodes of Ranvier are excitable regions of axonal membranes highly enriched in voltage-gated sodium channels that propagate action potentials. The mechanism of protein clustering at nodes has been a source of controversy. In this study, developmental analysis of nodes of Ranvier in optic nerve axons reveals that early node intermediates are defined by ankyrin-G. Other node components, including beta IV spectrin, voltage-gated sodium channels, and the L1 cell adhesion molecule neurofascin, are subsequently recruited to sites of ankyrin-G clustering. The role of intact paranodes in protein clustering was examined in the dysmyelinating mouse mutant jimpy. Jimpy mice do not have intact paranodal axoglial contacts, which is indicated by a complete lack of neurexin͞contactin-associated protein͞paranodin clustering in paranodes. In the absence of intact paranodes, ankyrin-G was still able to cluster, although fewer ankyrin clusters were seen in jimpy optic nerves than in wild-type optic nerves. Recruitment of Na v1.2, Nav1.6, beta IV spectrin, and neurofascin to sites of ankyrin-G clustering is unimpaired in jimpy mice, indicating that node formation occurs independent of intact paranodal axoglial contacts.