Opening the KcsA K+ Channel: Tryptophan Scanning and Complementation Analysis Lead to Mutants with Altered Gating
- 23 October 2002
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 41 (46), 13653-13662
- https://doi.org/10.1021/bi026393r
Abstract
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.Keywords
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