Analysis of Novel Mechanosensitive Channel Activities in Escherichia Coli

Abstract

Bacterial mechanosensitive channels gate in response to rapid increases in membrane tension that occur when cells transfer from high to low osmolarity environments. They open large, non-specific pores in the cytoplasmic membrane, releasing small solutes and ions to halt the instantaneous water influx that would otherwise cause cell lysis. Two major mechanosensitive channel families exist in bacteria (MscL and MscS) and protein databases show that Escherichia coli possess one MscL and six MscS family members. MscL was identified in E. coli in 1994 and our group has since cloned three of the MscS homologues: MscS, MscK and YbdG. These channels have been extensively studied and patch-clamp analysis on membranes of giant protoplasts shows that each gates at different levels of pressure and is associated with a specific conductance. MscL and MscS activities appear frequently in recordings and MscK is also regularly seen. Activities associated with YbdG protein expression are more elusive. However, during electrophysiological recordings additional openings are occasionally observed. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we use E. coli deletion strains and a modified protoplast preparation protocol to investigate previously uncharacterised mechanosensitive activities in E. coli using patch-clamp. When mscL mscS mscK ybdG null mutants are tested, infrequent mechanosensitive openings detected can be placed into three categories based on current amplitude and open-dwell properties. Incubation of cells in 0.5M NaCl during protoplast formation increases the probability of mechanosensitive activity in a patch. Furthermore, a new mechanosensitive channel conductance (∼1nS) is revealed in ∼40% of patches. Tests so far indicate that 0.5M KCl incubations do not cause the same effect. Further analysis of these novel activities will enhance our knowledge of mechanosensitive channels in bacteria.