Opening the KcsA K+ channel: tryptophan scanning and complementation analysis lead to mutants with altered gating.
The properties of the KcsA channel were investigated using a combination of tryptophan scanning of the two transmembrane helices followed by random mutagenesis at targeted residues. The tryptophan mutants were subjected to two screens: oligomeric stability and ability to complement the K+ uptake deficiency of the TK2420 Escherichia coli strain. Oligomeric stability is affected primarily by mutations at sites that border on and interact with the selectivity filter, while the complementation assays identified residues at the crossing point of the inner helices. Sites identified by the complementation assay in the tryptophan screen were subjected to random mutagenesis and selection by complementation. We have found two mutants, A108S and A108T, which have dramatically increased open probability while retaining the basic property of oligomeric stability.
Irizarry
Stacey N
SN
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Kutluay
Esin
E
Drews
Gabriele
G
Hart
Sarah J
SJ
Heginbotham
Lise
L
D000818:Animals; D000917:Antibody Formation; D001426:Bacterial Proteins / Q000502:physiology*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D007477:Ions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014364:Tryptophan / Q000378:metabolism*
D001426:Bacterial Proteins; D007477:Ions; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D014364:Tryptophan; D011188:Potassium