Genetic selection of activatory mutations in KcsA.
The KcsA potassium channel from Streptomyces lividans is one of the most actively studied ion channels. However, there are still unresolved issues about its gating mechanism in vivo because the channel is only activated by highly acidic intracellular pH, meaning that it will be mostly inactive in its host environment. In this study we have used a genetic complementation assay of K+-auxotrophic E. coli (TK2420) and S. cerevisiae (SGY1528) to identify activatory or 'gain-of-function' mutations which allow functional activity of KcsA in the physiological environment of two markedly different expression systems. These mutations clustered at the helix-bundle-crossing in both TM1 and TM2 (residues H25, L105, A108, T112, W113, F114, E118 and Q119), and include residues previously implicated in the pH-gating mechanism. We discuss how these gain-of-function mutations may result in their activatory phenotype, the relative merits of the E. coli and S. cerevisiae genetic complementation approaches for the identification of gating mutations in prokaryotic K+ channels, and ways in which this assay may be improved for future use in screening protocols.
Paynter
Jennifer J
JJ
Oxford Centre for Gene Function, Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK.
Sarkies
Peter
P
Andres-Enguix
Isabelle
I
Tucker
Stephen J
SJ
D001426:Bacterial Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000502:physiology*; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated; D048170:Streptomyces lividans
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C504448:KcsA protein, Streptomyces coelicolor; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C107790:prokaryotic potassium channel
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