GCN4 enhances the stability of the pore domain of potassium channel KcsA

FEBS J. 2008 Dec;275(24):6228-36. doi: 10.1111/j.1742-4658.2008.06747.x. Epub 2008 Nov 7.

Abstract

The prokaryotic potassium channel from Streptomyces lividans, KcsA, is the first channel that has a known crystal structure of the transmembrane domain. The crystal structure of its soluble C-terminal domain, however, still remains elusive. Biophysical and electrophysiological studies have previously implicated the essential roles of the C-terminal domain in pH sensing and in vivo channel assembly. We examined this functional assignment by replacing the C-terminal domain with an artificial tetramerization domain, GCN4-LI. The expression of KcsA is completely abolished when its C-terminal domain is deleted, but it can be rescued by fusion with GCN4-LI. The secondary and quaternary structures of the hybrid channel are very similar to those of the wild-type channel according to CD and gel-filtration analyses. The thermostability of the hybrid channel at pH 8 is similar to that of the wild-type but is insensitive to pH changes. This supports the notion that the pH sensor of KcsA is located in the C-terminal domain. The result obtained in the present study is in agreement with the proposed functions of the C-terminal domain and we show that the channel assembly role of the C-terminal domain can be substituted with a non-native tetrameric motif. Because tetramerization domains are found in different families of potassium channels and their presence often enhances the expression of channels, replacement of the elusive C-terminal domains with a known tetrameric scaffold could potentially assist the expression of other potassium channels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / physiology*
  • Basic-Leucine Zipper Transcription Factors
  • Chromatography, Gel
  • Chymotrypsin
  • DNA-Binding Proteins / chemistry
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Molecular
  • Potassium Channels / chemistry
  • Potassium Channels / physiology*
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Protein Stability
  • Saccharomyces cerevisiae Proteins / chemistry
  • Streptomyces lividans / physiology*
  • Thermodynamics
  • Transcription Factors / chemistry

Substances

  • Bacterial Proteins
  • Basic-Leucine Zipper Transcription Factors
  • DNA-Binding Proteins
  • GCN4 protein, S cerevisiae
  • Potassium Channels
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • prokaryotic potassium channel
  • Chymotrypsin