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Loss-of-Function Mutations at the Rim of the Funnel of Mechanosensitive Channel MscL

Kenjiro Yoshimura ^* , Takeshi Nomura  and Masahiro Sokabe  

^* Institute of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan; Cell-Mechanosensing Project, ICORP, Japan Science and Technology Agency, Nagoya 464-0856, Japan; and Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

Correspondence: Address reprint requests to Kenjiro Yoshimura, Institute of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan. Tel.: +81-29-853-6658; Fax: +81-29-853-6614; E-mail: kenjiro{at}biol.tsukuba.ac.jp.

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.

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