Sodium Channel β1 Subunits Promote Neurite Outgrowth in Cerebellar Granule Neurons

Tigwa H Davis,Chunling Chen,Lori L Isom

doi:10.1074/jbc.m410830200

Tigwa H Davis, Chunling Chen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m410830200

Copy DOI

Abstract

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.

Highlights

Intercellular communications mediate critical developmental events in neurons
We demonstrate that homophilic interactions between voltage-gated sodium channel ␤1 subunits promote neurite extension in cerebellar granule neurons
Based on structural and amino acid homologies, ␤ subunits are IGSF CAMs [11]. ␤1 and ␤2 exhibit homophilic and heterophilic adhesion and interact with extracellular matrix molecules [12,13,14,15,16]. ␤1- and ␤2-mediated homophilic cell adhesion results in recruitment of ankyrin to points of cell-cell contact, which in ␤1 can be inhibited by phosphorylation of a single, intracellular tyrosine residue [12, 17]. ␤1 and ␤2 mRNAs are expressed as early as P1 in brain

Summary

EXPERIMENTAL PROCEDURES

Cerebellar Dissociation—␤1(ϩ/ϩ) and ␤1(Ϫ/Ϫ) mice were generated and maintained as previously described, in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals [20]. Hybridization and detection was as previously described [20] These results were confirmed by Western blot analysis with an anti-␤4 antibody The longest neurite from each of the first 50, randomly selected, isolated granule cells were measured in at least five experiments per condition This process of analysis eliminated user bias. Construction and Analysis of the Soluble ␤1 Subunit—A vector constructed to express an inducible, truncated form of the ␤1 subunit was prepared using PCR with pcDNA3.1.␤1 encoding the rat ␤1 subunit (GenBankTM accession code NM_017288) as template, forward primer: 5Ј-GGATCCTTGCGCGGCCATGGGGAC-3Ј, and reverse primer: 5ЈGGAATTCTCTGACACGATGGATGC-3Ј These primers introduced BamHI and EcoRI sites at the 5Ј and 3Ј ends, respectively, of the extracellular ␤1 domain.

RESULTS

Findings

DISCUSSION