Sodium Channel β1 Subunit-mediated Modulation of Nav1.2 Currents and Cell Surface Density Is Dependent on Interactions with Contactin and Ankyrin

Abstract

Voltage-gated sodium channels are composed of a pore-forming ␣ subunit and at least one auxiliary ␤ subunit. Both ␤1 and ␤2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, ␤1, but not ␤2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of ␤1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for ␤1 association with contactin, ankyrin, and Na v 1.2. ␤1/␤2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the ␤1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Na v 1.2 intracellular domains interact directly with ankyrin G . We show that native Na v 1.2 associates with ankyrin G in cells in the absence of ␤ subunits and that this interaction is enhanced in the presence of ␤1 but not ␤1Y181E, a mutant that does not interact with ankyrin G . ␤1Y181E does not modulate Na v 1.2 channel function despite efficient association with Na v 1.2 and contactin. ␤1Y181E increases Na v 1.2 cell surface expression, but not as efficiently as wild type ␤1. ␤1/␤2 chimeras exchanging various regions of the ␤1 Ig loop were all ineffective in increasing Na v 1.2 cell surface density. Our results demonstrate that full-length ␤1 is required for channel modulation and enhancement of sodium channel cell surface expression.Voltage-gated sodium channels consist of a pore-forming ␣ subunit and at least one ␤ subunit (1-4). ␤1, ␤1A, and ␤3 non-covalently associate with ␣, whereas ␤2 and ␤4 are disulfide-linked to ␣ (4 -7). ␤ subunits have been shown to modulate channel kinetics and regulate cell surface expression of ␣ subunits (5, 8).Sodium channel ␤ subunits are unique among voltage-gated ion channel auxiliary subunits in that they also function as cell adhesion molecules (9). ␤1 and ␤2 interact with tenascin-C and tenascin-R, resulting in cellular repulsion (10, 11), and participate in homophilic (␤1-␤1 or ␤2-␤2) cell adhesion resulting in ankyrin recruitment to the plasma membrane at points of cell-cell contact (12). Ankyrin recruitment by ␤1 is dependent on phosphorylation of an intracellular tyrosine residue, ␤1Y181. Phosphorylation of ␤1Y181, or mutation of the tyrosine to glutamate mimicking a phosphorylation state (␤1Y181E), inhibits ␤1-mediated ankyrin recruitment (13) and results in ␤1 targeting to ankyrin-independent subcellular domains (14). ␤ subunits also participate in heterophilic celladhesive interactions with other Ig superfamily cell adhesion molecules. ␤1 interacts with contactin in vitro, resulting in increased Na v 1.2 cell surface expression without affecting channel function (15). Both ␤1 and ␤3 interact with neurofascin-186 (16), and the intracellular domain of ␤1 has been shown to interact with the receptor protein-tyrosine phosphatase ␤ (17).The purpose of the present stu...

Heterophilic Interactions of Sodium Channel β1 Subunits with Axonal and Glial Cell Adhesion Molecules

“…Antibodies and Constructs-Rabbit polyclonal antisera to sodium channel ␤1 and ␤2 subunits were described previously (19,22,24). A polyclonal anti-␤3 antibody was a gift from Dr. William A. Catterall (University of Washington, Seattle, WA) and has been described previously (20).…”

“…The Cn and Cn Ig -Fc constructs were gifts from Dr. Genviève Rougon (Laboratoire de Génétique et Physiologie du Dével-oppement, Marseille, France) (37). cDNAs encoding ␤1 and ␤2 were described previously (24). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes, Inc. (Eugene, OR).…”

“…Coexpression of ␤2 or ␤3 with Na v 1.2 in the absence of ␤1 resulted in no significant increases in sodium channel cell surface density over ␣ alone, whereas the presence of both contactin and ␤1 increased Na v 1.2 at the cell surface by 6-fold (17,24). We next investigated whether Nf186, Nf155, or NrCAM affected Na v 1.2 cell surface expression levels.…”

“…The association of contactin with sodium channels in vitro is dependent on the presence of ␤1, and this interaction results in increased sodium channel ␣ subunit expression at the cell surface (17). The interaction between ␤1 and contactin requires the Ig loop domain but not the juxtamembrane, transmembrane, or intracellular domains of ␤1 (24). We further investigated this interaction by examining the regions of contactin that are important for interaction with ␤1.…”

“…Nf186, Nr-CAM, ␤1, and ␤2 each interact in vitro with a key cytoskeletal anchoring protein, ankyrin G , that is also localized to nodes of Ranvier (21)(22)(23). ␤1-or ␤2-mediated homophilic cell-adhesive interactions result in ankyrin recruitment in Drosophila S2 cells, and the interaction of sodium channel ␣ subunits with ankyrin G is greatly enhanced in the presence of ␤1 subunits in vitro (22,24). We propose that the sodium channel signaling complex at nodes of Ranvier includes the CAMs contactin, NrCAM, and Nf186 as well as ankyrin G and that sodium channels interact with these proteins via ␤1 subunits.…”