Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.

Sodium channel β subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. β1 and β2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of β1 may be important for ankyrin recruitment and tested our hypothesis using β1 mutants replacing Tyr¹⁸¹ with alanine (β1Y181A), phenylalanine (β1Y181F), or glutamate (β1Y181E), or a truncated construct deleting all residues beyond Tyr¹⁸¹(β1L182_STOP). Ankyrin recruitment was observed in β1L182_STOP, showing that residues Ile¹⁶⁶-Tyr¹⁸¹ contain the major ankyrin recruiting activity of β1. Ankyrin recruitment was abolished in β1Y181E, suggesting that tyrosine phosphorylation of β1 may inhibit β1-ankyrin interactions. Ankyrin_G and β1 associate in rat brain membranes and in transfected cells expressing β1 and ankyrin_G in the absence of sodium channel α subunits. β1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. β1 and ankryin_G association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin_G and β1Y181E do not associate even in the absence of fibroblast growth factor treatment. β1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of β1Y181 may be a critical regulatory step in these developmental processes.

Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 ( DIAPH3 ) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy ( AUNA1 ), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3 . DNA sequencing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly conserved region of the 5′ UTR. The c.-172G > A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (≈1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G > A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK , are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.