In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv.
Chlorella virus-encoded membrane protein Kcv represents a new class of potassium channel. This 94-amino acids miniature K(+) channel consists of two trans-membrane alpha-helix domains intermediated by a pore domain that contains a highly conserved K(+) selectivity filter. Therefore, as an archetypal K(+) channel, the study of Kcv may yield valuable insights into the structure-function relationships underlying this important class of ion channel. Here, we report a series of new properties of Kcv. We first verified Kcv can be synthesized in vitro. By co-synthesis and assembly of wild-type and the tagged version of Kcv, we were able to demonstrate a tetrameric stoichiometry, a molecular structure adopted by all known K(+) channels. Most notably, the tetrameric Kcv complex retains its functional integrity in SDS (strong detergent)-containing solutions, a useful feature that allows for direct purification of protein from polyacrylamide gel. Once purified, the tetramer can form single potassium-selective ion channels in a lipid bilayer with functions consistent to the heterologously expressed Kcv. These finding suggest that the synthetic Kcv can serve as a model of virus-encoded K(+) channels; and its newly identified properties can be applied to the future study on structure-determined mechanisms such as K(+) channel functional stoichiometry.
Shim
Ji Wook
JW
Department of Biological Engineering, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
Yang
Mingming
M
Gu
Li-Qun
LQ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D008564:Membrane Potentials; D017862:Phycodnaviridae / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D004279:DNA, Viral; C405463:Kcv potassium channel, Chlorella virus; D015221:Potassium Channels; D011994:Recombinant Proteins; D014764:Viral Proteins