Gating-associated conformational changes in the mechanosensitive channel MscL.
Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3-4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patch-clamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, Ca(2+) influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically cross-linked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation.
Yoshimura
Kenjiro
K
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan. kenjiro@biol.tsukuba.ac.jp
Usukura
Jiro
J
Sokabe
Masahiro
M
D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D004586:Electrophoresis; D004594:Electrophysiology; D015640:Ion Channel Gating; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008081:Liposomes; D040542:Mechanotransduction, Cellular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017384:Sequence Deletion
D007473:Ion Channels; D008081:Liposomes; D050505:Mutant Proteins; D002118:Calcium