Identification of amino acid residues in fibroblast growth factor 14 (FGF14) required for structure-function interactions with voltage-gated sodium channel Nav1.6

Syed R. Ali, Aditya K. Singh, Fernanda Laezza

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 FGF14V160A or the FGF14K74A/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 FGF14V160A to the Nav1.6 C-tail compared with FGF14K74A/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.

Original languageEnglish (US)
Pages (from-to)11268-11284
Number of pages17
JournalJournal of Biological Chemistry
Volume291
Issue number21
DOIs
StatePublished - May 20 2016

Fingerprint

NAV1.6 Voltage-Gated Sodium Channel
Amino Acids
Dimers
Mutation
Proteins
fibroblast growth factor 14
Alanine

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{e5f789b76b8f47c8bd71203bdac99e9f,
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 FGF14V160A or the FGF14K74A/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 FGF14V160A to the Nav1.6 C-tail compared with FGF14K74A/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.",
author = "Ali, {Syed R.} and Singh, {Aditya K.} and Fernanda Laezza",
year = "2016",
month = "5",
day = "20",
doi = "10.1074/jbc.M115.703868",
language = "English (US)",
volume = "291",
pages = "11268--11284",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "21",

}

TY - JOUR

T1 - Identification of amino acid residues in fibroblast growth factor 14 (FGF14) required for structure-function interactions with voltage-gated sodium channel Nav1.6

AU - Ali, Syed R.

AU - Singh, Aditya K.

AU - Laezza, Fernanda

PY - 2016/5/20

Y1 - 2016/5/20

N2 - 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 FGF14V160A or the FGF14K74A/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 FGF14V160A to the Nav1.6 C-tail compared with FGF14K74A/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.

AB - 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 FGF14V160A or the FGF14K74A/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 FGF14V160A to the Nav1.6 C-tail compared with FGF14K74A/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.

UR - http://www.scopus.com/inward/record.url?scp=84971320836&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84971320836&partnerID=8YFLogxK

U2 - 10.1074/jbc.M115.703868

DO - 10.1074/jbc.M115.703868

M3 - Article

VL - 291

SP - 11268

EP - 11284

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -