Modulation of the voltage-gated potassium channel (Kv4.3) and the auxiliary protein (KChIP3) interactions by the current activator NS5806

J Biol Chem. 2014 Nov 14;289(46):32201-32213. doi: 10.1074/jbc.M114.577528. Epub 2014 Sep 16.

Abstract

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2-5 μM in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μM and 2.7 ± 0.1 μM in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (Kd = 1.9 ± 0.1 μM) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of KV4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.

Keywords: Calcium-binding Protein; Calsenilin; Fluorescence Resonance Energy Transfer (FRET); Isothermal Titration Calorimetry (ITC); KChIP; Kv4; NCS; NS5806; Potassium Channel; Protein Drug Interaction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Anisotropy
  • Binding Sites
  • Calcium / metabolism
  • Calcium-Binding Proteins / metabolism
  • Calorimetry
  • Fluorescence Resonance Energy Transfer
  • Kv Channel-Interacting Proteins / metabolism*
  • Magnesium / chemistry
  • Mice
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Phenylurea Compounds / chemistry*
  • Protein Binding
  • Protein Structure, Tertiary
  • Repressor Proteins / metabolism*
  • Shal Potassium Channels / metabolism*
  • Spectrometry, Fluorescence
  • Tetrazoles / chemistry*

Substances

  • 1-(3,5-bis-trifluoromethylphenyl)-3-(2,4-dibromo-6-(1H-tetrazol-5-yl)phenyl)urea
  • Calcium-Binding Proteins
  • Csen protein, mouse
  • Kv Channel-Interacting Proteins
  • Phenylurea Compounds
  • Repressor Proteins
  • Shal Potassium Channels
  • Tetrazoles
  • Magnesium
  • Calcium