Key result
Random mutagenesis of the KcsA potassium channel identified novel activatory mutations clustered at the helix-bundle crossing that permit functional activity in E. coli and S. cerevisiae.
This study identifies specific gain-of-function mutations in the KcsA potassium channel that enable its activation at physiological pH, providing insights into its gating mechanism.
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.
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Paynter et al. (2008) studied this question. Random mutagenesis of KcsA vs. Wild-type KcsA was evaluated on Complementation of K+-uptake deficiency in E. coli and S. cerevisiae. Random mutagenesis of the KcsA potassium channel identified novel activatory mutations clustered at the helix-bundle crossing that permit functional activity in E. coli and S. cerevisiae.
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