Genetic Screen for Potassium Leaky Small Mechanosensitive Channels (MscS) in Escherichia coli

MscS is a bacterial mechanosensitive channel (MS) that opens upon membrane stretch and will jettison osmolytes protecting the bacteria (together with another MS channel MscL) against lysis during severe osmotic downshocks. Its transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain which changes its conformation upon gating but its involvement in gating, although evident from earlier studies, is not understood.
In this paper we screened MscS for mutations that result in leaky channels and as a result rescue growth of E. coli strains deficient in potassium transport systems on potassium limiting media. We have been able to isolate a plethora of mutations that cause MscS to leak potassium. After phenotypic analysis, we have also been able to assign the role of several of them to the particular functional state of the channel.
Except of confirming earlier findings on the role of transmembrane domains (TM1, TM2, TM3) in gating, we found that there is a transient interaction of the pore forming domain (TM3) and the cytoplasmic domain of the channel (the beta domain). TM3 and beta dissociate upon channel opening while their attachment maintains the channel shut conformation. The fragments of TM3 and beta participating in the interaction are highly conserved among MscS homologs what indicates evolutionary pressure against leaky channels.

Date of publication
2 March 2011