Tetrameric stoichiometry of a prokaryotic K+ channel.
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.
Heginbotham
L
L
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts.
Odessey
E
E
Miller
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001704:Biopolymers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D013302:Streptomyces / Q000737:chemistry
D001426:Bacterial Proteins; D001704:Biopolymers; D015221:Potassium Channels; C107790:prokaryotic potassium channel