Voltage-gated sodium channels are comprised of a pore-forming alpha-subunit and one or more auxiliary subunits (from the gene family containing beta1, beta2, beta3, and beta1A). Sodium channels are localized at high density to the central nervous system (CNS) nodes of Ranvier of myelinated axons. The overall goal of this research is to define the molecular composition of the sodium channel signaling complex present at the nodes of Ranvier. We propose that contactin is an important protein in regulating the localization and function of voltage-gated sodium channels in the CNS. The first specific aim of this study is to determine the domains of the sodium channel beta1 subunit that are responsible for interaction with contactin. This will be accomplished by making chimeras between beta1 and beta2 by exchanging their Ig loops, the proposed site of interaction. Since beta2 does not interact with contactin, a beta2 chimera with a beta1 Ig loop should gain the ability to interact with contactin. From here, the specific region of the beta1 Ig loops necessary for interaction with contactin can elucidated by making more chimeras. To determine whether heterophilic interactions between beta1 subunits and contactin cause aggregation in Drosophila S2 cells resulting in the recruitment of ankyrin to points of cell-cell contact, an S2 cell line transfected with contactin will be mixed with another transfected with beta1. By using a vital fluorescent membrane dye, DiI, and labeling only one of the transfected cell lines, aggregates can be examined for heterophilic interactions between contactin and beta1. Further, aggregates exhibiting heterophilic interactions can be probed with an anti-ankyrin antibody to look at the ability of the interactions to recruit ankyrin to points of cell-cell contact. The third specific aim of the study is to determine whether interaction of contactin with the beta1 subunit modulates tyrosine phosphorylation of the beta1 subunits. We will determine the phosphorylation state of beta1 by incubating cell lines stably expressing beta1 with a soluble contactin molecule and looking for altered phosphorylation. The completion of these studies will give insight into development of mature nodes of Ranvier, as well as a possible mechanism for the anchoring of sodium channels to the actin cytoskeleton.
