title: Identification of Amino Acid Residues in Fibroblast Growth Factor 14 (FGF14) Required for Structure-Function Interactions with Voltage-gated Sodium Channel Nav1.6.
abstract: The voltage-gated Na(+) (Nav) channel provides the basis for electrical excitability in the brain. This channel is regulated by a number of accessory proteins including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF family. In addition to forming homodimers, FGF14 binds directly to the Nav1.6 channel C-tail, regulating channel gating and expression, properties that are required for intrinsic excitability in neurons. Seeking amino acid residues with unique roles at the protein-protein interaction interface (PPI) of FGF14·Nav1.6, we engineered model-guided mutations of FGF14 and validated their impact on the FGF14·Nav1.6 complex and the FGF14:FGF14 dimer formation using a luciferase assay. Divergence was found in the β-9 sheet of FGF14 where an alanine (Ala) mutation of Val-160 impaired binding to Nav1.6 but had no effect on FGF14:FGF14 dimer formation. Additional analysis revealed also a key role of residues Lys-74/Ile-76 at the N-terminal of FGF14 in the FGF14·Nav1.6 complex and FGF14:FGF14 dimer formation. Using whole-cell patch clamp electrophysiology, we demonstrated that either the FGF14(V160A) or the FGF14(K74A/I76A) mutation was sufficient to abolish the FGF14-dependent regulation of peak transient Na(+) currents and the voltage-dependent activation and steady-state inactivation of Nav1.6; but only V160A with a concomitant alanine mutation at Tyr-158 could impede FGF14-dependent modulation of the channel fast inactivation. Intrinsic fluorescence spectroscopy of purified proteins confirmed a stronger binding reduction of FGF14(V160A) to the Nav1.6 C-tail compared with FGF14(K74A/I76A) Altogether these studies indicate that the β-9 sheet and the N terminus of FGF14 are well positioned targets for drug development of PPI-based allosteric modulators of Nav channels.
authors.affiliation: From the Department of Pharmacology and Toxicology, the Pharmacology and Toxicology Graduate Program.
authors.forename: Syed R
authors.initials: SR
authors.lastname: Ali
authors.affiliation: From the Department of Pharmacology and Toxicology.
authors.forename: Aditya K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: From the Department of Pharmacology and Toxicology, the Mitchell Center for Neurodegenerative Diseases, the Center for Addiction Research, the Center for Environmental Toxicology, and the Center for Biomedical Engineering, University of Texas Medical Branch, Galveston, Texas 77555 felaezza@utmb.edu.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Laezza
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D005346:Fibroblast Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D062557:NAV1.6 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D062557:NAV1.6 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C100681:SCN8A protein, human; C112971:fibroblast growth factor 14; D005346:Fibroblast Growth Factors
keywords: FGF14; Nav1.6; amino acid; electrophysiology; fibroblast growth factor (FGF); hot spots; ion channel; protein-protein interaction; split-luciferase assay; voltage-gated sodium channels