Pore mutations of the Escherichia coli MscS channel affect desensitisation but not ionic preference

¹ University of Aberdeen

^* To whom correspondence should be addressed. E-mail: m.d.edwards{at}abdn.ac.uk.

Submitted on October 9, 2007
Revised on October 30, 2007
Accepted on 28 November 2007

	Abstract

Mechanosensitive channels rescue bacterial cells from a fate of lysis when they transfer from a high to low osmolarity environment. Of three Escherichia coli mechanosensitive proteins studied to date, only MscS-Ec demonstrates a small anionic preference and a desensitised, non-conducting state under sustained pressure. Little is known about the mechanisms generating these distinctive properties. Eliminating the sole positive charge in the MscS-Ec pore region (Arg88) did not alter anionic preference. Adding positive charges at either end of the pore did not augment anionic preference and placing negative charges within the pore did not diminish it. Thus, pore charges do not control this characteristic. However, from this analysis we identified mutations at the hinge residue of the MscS-Ec pore helix (Gly113) that profoundly affected ability of the channel to desensitise. Substitution with non-polar (Ala; Pro) or polar (Asp; Arg; Ser) residues inhibited transition to the desensitised state. Interestingly, Gly113 replaced with Met did not impede desensitisation. Thus, although Gly is not specifically required at position 113, MscS desensitisation is strongly influenced by the residue situated here. Mutations at residues further into the pore also regulated desensitisation. Transition to this unique mechanosensitive channel state is discussed in terms of the existing data.

Key Words: electrophysiology, inactivation, ionic selectivity, mechanosensitive, membrane protein