General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K(+) channel

J Biol Chem. 2000 Feb 18;275(7):4928-36. doi: 10.1074/jbc.275.7.4928.

Abstract

The structural bases of general anesthetic action on a neuronal K(+) channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K(+) channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Butanol / pharmacology
  • Amino Acid Sequence
  • Anesthetics, General / pharmacology*
  • Animals
  • Cytoplasm / drug effects*
  • Cytoplasm / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neurons / drug effects*
  • Neurons / metabolism
  • Potassium Channels / chemistry
  • Potassium Channels / drug effects*
  • Potassium Channels, Voltage-Gated*
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / drug effects
  • Sequence Homology, Amino Acid
  • Shaw Potassium Channels
  • Xenopus
  • Xenopus Proteins*

Substances

  • Anesthetics, General
  • KCNC4 protein, Xenopus
  • Potassium Channels
  • Potassium Channels, Voltage-Gated
  • Recombinant Proteins
  • Shaw Potassium Channels
  • Xenopus Proteins
  • 1-Butanol