Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA.
All K(+)-channels are stabilized by K(+)-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K(+)-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K(+)-channel.
Raja
Mobeen
M
Institute for Biophysics, Johannes Kepler University Linz, 4040 Linz, Austria. mraja@pmcol.ualberta.ca
Vales
Elisabeth
E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011336:Probability; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C107790:prokaryotic potassium channel; D014364:Tryptophan