Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

Abstract

4Corresponding author P.Blount and S.I.Sukharev contributed equally to this studyWe have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na + /K + translocating AtHKT1 protein, a member of the superfamily of K + transporters

“…3). When the PhoA domain of the fusion protein remains in the cytoplasm, it does not form the intrachain disulfide bond essential for activity (24), and it becomes sensitive to proteolytic degradation (24,32), as has been reported for PhoA-fusion experiments with other ion transporters (13,25,33). Some of the degradation may have occurred after the cells were broken for making membranes because all of the intact fusion proteins were detected in samples taken from growing E. coli cells (results not shown).…”

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

“…4). Early modelling of the MscL monomer directed a programme of mutagenesis that targeted these same regions (Blount et al, 1996a). Small lesions, such as point mutations, in these regions yielded mutants with altered characteristics, indicating that these regions are crucial to the normal functioning of this channel.…”

“…Single-channel analysis E. coli giant spheroplasts were generated and used in patch clamp experiments as described previously (Martinac et al, 1987), with modifications (Sukharev et al, 1994;Blount et al, 1996a). Briefly, excised, inside-out patches were examined at room temperature under symmetrical conditions using a buffer consisting of 200 mM KCl, 90 mM MgCl 2 , 10 mM CaCl 2 and 5 mM HEPES adjusted to pH 6.0.…”

“…A piezoelectric pressure transducer (Micro Switch), calibrated with a solid-state pneumatic gauge (Bio-Tec), was used to measure the pressure response of the channels. The pressure threshold response of the MscL homologues was determined with respect to MscS as described previously (Blount et al, 1996a). The pressure threshold for actuation of the MscL channel, with respect to the actuation threshold of MscS, was determined at a P o of approximately 0.0003 to 0.0075 and within 5 s or less of pressure application to avoid desensitization of MscS.…”

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

“…Its 3D structure, unable to be modeled in the first MscL crystal structure reported (24), was revealed in the reinterpreted structure as a single a-helix (139). Several functional studies have demonstrated that even small N-terminal deletions or changes to the N-terminal amino-acid sequence are poorly tolerated, resulting in nonfunctional channels or channels which exhibit altered pressure sensitivity (20,59,143). More recent results suggest that the N-terminus contributes to MscL mechanosensitivity by directly sensing membrane tension and transferring it to the pore lining helices, most likely by maintaining its position along the membranecytoplasm interface (31,68).…”