A molecular analysis of the glutathione-gated potassium channel, KefB, in Escherichia coli
The potassium efflux systems KefB and KefC may be targets for novel antibiotics. The kefC gene has been cloned and molecular analysis on the KefC protein has begun. The yabF gene, which lies directly upstream of the kefC gene on the E. coli chromosome, is required for KefC activity. The aims of the project were to characterise the physiological role of KefB, to clone the KefB gene, and to initiate molecular studies on the regulation of KefB activity. In the course of these studies, it has been shown that; 1. KefB and KefC do not afford protection against all electrophiles since they do not afford protection against the electrophile iodoacetate; 2. KefB and KefC can be partially activated by electrophiles, without the formation of a glutathione-adduct. However, complete activation of KefB and KefC requires the formation of a glutathione-adduct; 3. There may be more than one site for direct activation of KefB and KefC; a periplasmic and a cytoplasmic site; 4. The KefB protein is homologous to the KefC protein, the amino-terminal domain spans the membrane between six and twelve times and the carboxy-terminal domain is cytoplasmic; 5. KefB, for full activation, requires the ancillary protein YhaH, a YabF homologue. YhaH and YabF are involved in the regulation of KefB (and possibly KefC), but, for activation, each is specific for its own system, i.e. only YhaH activates KefB and only YabF activates KefC. Based upon this work, an updated model for the different states of KefB and KefC has been proposed, the involvement of the YhaH and YabF proteins are discussed and molecular studies on KefB have been initiated.