Structural requirements for interaction of sodium channel β1 subunits with ankyrin

Sodium channel beta subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. beta1 and beta2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of beta1 may be important for ankyrin recruitment and tested our hypothesis using beta1 mutants replacing Tyr(181) with alanine (beta1Y181A), phenylalanine (beta1Y181F), or glutamate (beta1Y181E), or a truncated construct deleting all residues beyond Tyr(181) (beta1L182(STOP)). Ankyrin recruitment was observed in beta1L182(STOP), showing that residues Ile(166)-Tyr(181) contain the major ankyrin recruiting activity of beta1. Ankyrin recruitment was abolished in beta1Y181E, suggesting that tyrosine phosphorylation of beta1 may inhibit beta1-ankyrin interactions. Ankyrin(G) and beta1 associate in rat brain membranes and in transfected cells expressing beta1 and ankyrin(G) in the absence of sodium channel alpha subunits. beta1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. beta1 and ankryin(G) association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin(G) and beta1Y181E do not associate even in the absence of fibroblast growth factor treatment. beta1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of beta1Y181 may be a critical regulatory step in these developmental processes.