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Interaction between the Cytoplasmic and Transmembrane Domains of the Mechanosensitive Channel MscS

Takeshi Nomura ^*, Masahiro Sokabe ^*   and Kenjiro Yoshimura ^*  ¶

^* International Cooperative Research Project/Solution Oriented Research for Science and Technology, Cell Mechanosensing, Japan Science and Technology Agency, Nagoya 466-8550, Japan; Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; and ^¶ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan

Correspondence: Adddress reprint requests to Kenjiro Yoshimura, Structural Biosciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan. Tel.: 81-29-853-6658; Fax: 81-29-853-6614; E-mail: kenjiro{at}biol.tsukuba.ac.jp.

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.