Abstract:
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.
journal_name
Channels (Austin)journal_title
Channels (Austin, Tex.)authors
Paynter JJ,Sarkies P,Andres-Enguix I,Tucker SJdoi
10.4161/chan.2.6.6874subject
Has Abstractpub_date
2008-11-01 00:00:00pages
413-8issue
6eissn
1933-6950issn
1933-6969pii
6874journal_volume
2pub_type
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