Voltage-gated sodium channel associated proteins: Modulation of Nav1.6 by FHF2B andp38 MAP kinase

Voltage-gated sodium channel protein partners regulate channel trafficking and/or modulate the biophysical properties of the channels. A yeast two-hybrid screen identified FHF2B, a member of the fibroblast growth factor homologous factor family, as a partner of Nav1.6. FHF2 is abundantly expressed in the hippocampus and DRG neurons and co-localizes with Nav1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. Retinal ganglion cells and ventral horn motor neurons produce low levels of FHF2 and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. The co-expression of FHF2B and Nav1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The discovery of FHF2 at nodes of Ranvier in sensory axons, but not in motor axons, is the first published example of differential expression of a protein at nodes along functionally different types of axons, and the effect of this interaction on channel density may explain in part the differences between the biophysical properties of sensory and motor fibers.;We also examined the phosphorylation of Nav1.6 by p38, a member of the mitogen-activated protein kinase (MAPK) family. The rationale is that injury-induced activation of p38 leads to phosphorylation of Nav1.6, which regulates the sodium channel density or biophysical properties. Co-immunoprecipitation experiments from rat brain and transfected HEK293 cells confirmed the association of Nav1.6 and p38alpha. Kinase assays using purified active p38 kinase and GST-fusion proteins confirmed that the first and second intracellular loops of Nav1.6 are phosphorylated by p38. Electrophysiological analysis of ND7/23 cells transfected with Nav1.6 and treated with anisomycin to activate p38 show that activated p38 leads to a significant reduction in the current density. The p38 effect was prevented by the treatment of the cells with SB203580, a specific inhibitor of p38. This represents the first demonstration of Nav1.6 modulation by a kinase, and the first example of sodium channel regulation by a MAP kinase.