Gating-associated conformational changes in the mechanosensitive channel MscL

*Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan;
^†International Cooperative Research Project/Solution Oriented Research for Science and Technology, Cell-Mechanosensing Project, Japan Science and Technology Agency, 65 Tsurumai, Nagoya 466-8550, Japan;
^‡Department of Bioenvironmental Science, Okazaki Institute for Integrative Biosciences, Okazaki, Aichi 444-8787, Japan;
^¶Ecotopia Science Institute, Nagoya University, Nagoya 464-8603, Japan;
^‖Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; and
**Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan

Edited by Martin Chalfie, Columbia University, New York, NY, and approved January 14, 2008 (received for review October 4, 2007)

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. 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, 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.

Footnotes

^§To whom correspondence should be addressed:
Structural Bioscience, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan.
E-mail: kenjiro{at}biol.tsukuba.ac.jp
Author contributions: K.Y. and M.S. designed research; K.Y. and J.U. performed research; K.Y. and J.U. analyzed data; and K.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.