TY - JOUR
T1 - Chemically charging the pore constriction opens the mechanosensitive channel MscL
AU - Yoshimura, Kenjiro
AU - Batiza, Ann
AU - Kung, Ching
N1 - Funding Information:
This work was supported by National Institutes of Health grant GM47856 (C.K.) and by the Ministry of Education, Culture, Art and Sports of Japan (K.Y.).
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
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U2 - 10.1016/S0006-3495(01)76192-9
DO - 10.1016/S0006-3495(01)76192-9
M3 - Article
C2 - 11325722
AN - SCOPUS:0035035068
SN - 0006-3495
VL - 80
SP - 2198
EP - 2206
JO - Biophysical Journal
JF - Biophysical Journal
IS - 5
ER -