CK2 accumulation at the axon initial segment depends on sodium channel Nav1

•Phosphorylation of Nav1 ankyrin-binding motif occurs in vivo.•Nav1 expression is necessary for CK2 localization at the AIS.•CK2-Nav1 molecular complex modulates CK2-clustering at the AIS. Accumulation of voltage-gated sodium channel Nav1 at the axon initial segment (AIS), results from a direct interaction with ankyrin G. This interaction is regulated in vitro by the protein kinase CK2, which is also highly enriched at the AIS. Here, using phosphospecific antibodies and inhibition/depletion approaches, we showed that Nav1 channels are phosphorylated in vivo in their ankyrin-binding motif. Moreover, we observed that CK2 accumulation at the AIS depends on expression of Nav1 channels, with which CK2 forms tight complexes. Thus, the CK2–Nav1 interaction is likely to initiate an important regulatory mechanism to finely control Nav1 phosphorylation and, consequently, neuronal excitability.