Genetic screen for potassium leaky small mechanosensitive channels (MscS) in Escherichia coli: recognition of cytoplasmic β domain as a new gating element.
Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.
Koprowski
Piotr
P
Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Grajkowski
Wojciech
W
Isacoff
Ehud Y
EY
Kubalski
Andrzej
A
D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D055724:Electrophysiological Phenomena; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D016062:Porosity; D011188:Potassium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D011188:Potassium