NS1643 interacts around L529 of hERG to alter voltage sensor movement on the path to activation

Biophys J. 2015 Mar 24;108(6):1400-1413. doi: 10.1016/j.bpj.2014.12.055.

Abstract

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • Cresols / metabolism*
  • Cresols / pharmacology*
  • ERG1 Potassium Channel
  • Ether-A-Go-Go Potassium Channels / genetics
  • Ether-A-Go-Go Potassium Channels / metabolism*
  • Fluorometry
  • Humans
  • Membrane Potentials / physiology
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutation
  • Oocytes
  • Patch-Clamp Techniques
  • Phenylurea Compounds / metabolism*
  • Phenylurea Compounds / pharmacology*
  • Protein Structure, Secondary
  • Transfection
  • Xenopus laevis

Substances

  • Cresols
  • ERG1 Potassium Channel
  • Ether-A-Go-Go Potassium Channels
  • KCNH2 protein, human
  • Phenylurea Compounds
  • 1,3-bis(2-hydroxy-5-trifluoromethylphenyl)urea