Biophys J, May 2001, p. 2198-2206, Vol. 80, No. 5

Chemically Charging the Pore Constriction Opens the Mechanosensitive Channel MscL

Kenjiro Yoshimura,^* Ann Batiza, and Ching Kung

 ^*Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan; and  Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 USA

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (K. Yoshimura, A. Batiza, M. Schroeder, P. Blount, and C. Kung, 1999, Biophys. J. 77:1960-1972). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.

Biophys J, May 2001, p. 2198-2206, Vol. 80, No. 5
© 2001 by the Biophysical Society   0006-3495/01/05/2198/09  $2.00

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