Chemically charging the pore constriction opens the mechanosensitive channel MscL

Area of Science:

• Biophysics
• Molecular Biology
• Cell Biology

Background:

• Mechanosensitive channels like MscL are crucial for bacterial survival during osmotic stress.
• Previous studies indicated that pore-lining residues influence MscL channel gating.
• The hydrophobicity of substitutions at MscL's Gly-22 residue affects channel gating.

Purpose of the Study:

• To investigate the role of MscL's Gly-22 residue in channel gating using chemical modifications.
• To explore the environmental changes within the MscL pore during gating.
• To understand the gating barrier of mechanosensitive channels.

Main Methods:

• Site-directed mutagenesis to introduce a cysteine at position 22 (G22C) in MscL.
• Patch-clamp electrophysiology to record MscL channel activity.
• Chemical modification of the introduced cysteine with methanethiosulfonate (MTS) reagents of varying charges and polarities.

Main Results:

• Charged MTS reagents induced spontaneous MscL gating without mechanical tension.
• Polar and hydrophobic MTS reagents modulated the mechanical gating threshold.
• Cysteine 22 accessibility varied from cytoplasmic (open state) and periplasmic (closed state) sides, suggesting conformational changes.

Conclusions:

• MscL residue 22 is in a hydrophobic environment in the closed state and a hydrophilic environment in the open state.
• The transition to a hydrophilic environment during opening contributes to the gating barrier.
• These findings provide insights into the mechanism of mechanosensitive channel gating.