Gating-associated conformational changes in the mechanosensitive channel MscL

Kenjiro Yoshimura, Jiro Usukura, Masahiro Sokabe

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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. Patchclamp 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, Ca2+ 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.

Original languageEnglish
Pages (from-to)4033-4038
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number10
DOIs
Publication statusPublished - 2008 Mar 25

Keywords

  • Bacterial ion channel
  • Electon microscopy
  • Liposome
  • Low-angle rotary shadowing
  • Patch clamp

ASJC Scopus subject areas

  • General

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