The molecular basis of the ligand-gated potassium efflux system KEFC
Aspects of the molecular basis of potassium transport via the KefC potassium efflux system in E. coli were examined. Separate cloning of the proposed membrane-spanning and cytoplasmic domains of KefC was carried out. A KefC construct was created that contained a amber stop codon at residue 382, between the two domains. When cloned into a prokaryotic expression vector, transformed strains grew poorly. No activity was detected in either an amber suppressor strain, or a strain lacking an amber suppressor, in the presence or absence of IPTG. A series of strains possessing mutant kefC alleles that cause spontaneous potassium loss via KefC were studied. The mutations in the mutant kefC strains were identified as missense mutations, which mapped to two regions of KefC; a sequence of amino acids in the N-terminal domain, (HALESDIE), and the Rossman fold within the C-terminal, cytoplasmic, domain. Recently, it has been discovered that an upstream gene, yabF, which overlaps kefC by eight base pairs, is involved in KefC activity. The sequence of yabF, from Frag5 and the mutant strains was examined and was not mutated in any of the strains, but the sequence of yabF was different from that published by the E. coli genome sequencing project. Studies on kefB mutant strains were initiated. In vitro site-directed mutagenesis of cloned kefC was used to analyse the importance of two residues in the Rossman fold, R416 and S420. The KefCR416S mutations results in spontaneous potassium loss, whereas the KefCS420A mutation does not. The interaction of KefC subunits was confirmed. The R416S mutation, (in the Rossman fold), and the D264A mutation, (in the HALESDIE sequence), were co-dominant. Spontaneous potassium loss via KefCR416S was found to require YabF and glutathione.