Tetrameric stoichiometry of a prokaryotic K+ channel

Biochemistry. 1997 Aug 19;36(33):10335-42. doi: 10.1021/bi970988i.

Abstract

Genes with sequences reminiscent of neuronal K+ channels have recently been identified in prokaryotes. These putative K+ channels appear to be integral membrane proteins, with multiple transmembrane sequences identified by hydrophobicity analysis and a sequence strikingly similar to the pore-lining "P-region" motif found in all known eukaryotic K+ channels. This study examines the oligomeric state and stability in detergent micelles of SliK, a K+ channel homologue from Streptomyces lividans. A synthetic gene for SliK was expressed at high levels in Escherichia coli, and the protein was purified. The predominant form of the protein runs in SDS-PAGE gels as an oligomer of the 19-kDa polypeptide, but harsh treatments such as heat or high pH convert this slowly-migrating material into monomeric form. A "mass-tagging" strategy developed to examine subunit stoichiometry shows that SliK is a homotetramer in SDS and dodecyl maltoside micelles. The tetrameric structure can be disrupted by P-region mutations known to prevent the functional expression of neuronal K+ channels. The tetramer is remarkably stable, showing no conversion to the monomeric form after 14 days at room temperature. Although SliK-mediated cation flux activity was not observed, the tetrameric behavior of the protein argues that SliK may provide a system for a direct attack on the structure of a K+ channel P-region sequence.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins*
  • Base Sequence
  • Biopolymers
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Genes, Synthetic
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Potassium Channels / chemistry*
  • Potassium Channels / genetics
  • Streptomyces / chemistry

Substances

  • Bacterial Proteins
  • Biopolymers
  • Potassium Channels
  • prokaryotic potassium channel