Defining the Protein:Protein Interaction Interface of FGF14:Nav1.6 Complex

Abstract

The voltage-gated Na+ (Nav) channel is composed of transmembrane spanning domains and of a cytosolic C-terminal tail which regulates channel function through protein:protein interactions (PPI) with auxiliary proteins, including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF (iFGF) family. In addition to binding to the Nav C-tail, FGF14 forms homodimers and previous structural studies have proposed a conserved surface common for the Nav channel and the iFGF homodimer formation. Seeking for potential differences between the FGF14:Nav1.6-C-tail complex and the FGF14:FGF14 dimer interface, we have engineered model-based amino acid residue mutations at predicted FGF14 hot-spots and begun to screen for their impact on the protein complex stability. Using the in-cell split-luciferase complementation assay to reconstitute the FGF14:Nav1.6-C-tail and the FGF14:FGF14 complex, we identified a point of divergence at the FGF14V160 residue whose mutation led to opposite effects on the relative binding affinity to the FGF14 monomer versus the Nav1.6-C-tail. Functional studies using whole-cell patch-clamp electrophysiology indicated that V160 is a critical residue for FGF14 modulation of Nav1.6-mediated currents that can be abolished by Ala mutation. Initial studies using intrinsic fluorescence showed efficient interaction between purified FGF14 and the Nav1.6 C-tail. Surface plasmon resonance and isothermal titration calorimetry measurements to evaluate the role of V160 in regulating binding affinity of FGF14 to the Nav1.6 C-tail are underway. With its unique role in the regulating the FGF14 binding and function, the V160 residue is well-positioned as target site for PPI-based drug development against Nav channels.Supported by: R01MH095995 (FL) and the Gulf Coast Consortia NIGMS Grant No.1 T32 GM089657-04 (SRA).