Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA.
Four subunits of the bacterial Streptomyces lividans protein KcsA form a K+ channel which can be functionally reconstituted in vitro. Here we show that substitution of the tyrosine residue 82 by cysteine, valine or threonine, but not by glycine, led to functional channel types. Like the wild-type (WT) and an L81C channel, the mutant channels exhibit an internal pH-sensitive side and are cation selective. Based on the relative positions of the blocker tetraethylammonium within the electric field, the external entryways of the channels are concluded to have similar dimensions. For inward currents, the WT and the mutant channels vary in the occupancy of their subconductance states and concomitantly in their mean currents. Rectification properties are scarcely (L81C), little (Y82C) or considerably (Y82T and Y82V) altered. The data suggest that the amino acid type in position 82 stabilizes to varying degrees an open conformation within the external region of the permeation pathway.
Meuser
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D
Angewandte Genetik der Mikroorganismen, FB Biologie/Chemie, Universität Osnabrück, Germany.
Splitt
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H
Wagner
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R
Schrempf
H
H
D001426:Bacterial Proteins; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004594:Electrophysiology; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000378:metabolism; D013696:Temperature; D019789:Tetraethylammonium / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014633:Valine / Q000737:chemistry
D001426:Bacterial Proteins; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D013912:Threonine; D019789:Tetraethylammonium; D014633:Valine; D003545:Cysteine; D005998:Glycine