Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS
Takeshi Nomura1, Masahiro Sokabe, Kenjiro Yoshimura
1International Cooperative Research Project/Solution Oriented Research for Science and Technology, Cell Mechanosensing, Japan Science and Technology Agency, Nagoya 466-8550, Japan.
Abstract:
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.
Insights
Specific charged residues in the bacterial mechanosensitive channel MscS, particularly Asp-62 and Arg-131, are crucial for channel gating and inactivation. Their electrostatic interactions regulate MscS function during hypoosmotic shock.
Area of Science:
- Molecular biology
- Biophysics
- Cellular biology
Background:
- The bacterial mechanosensitive channel MscS (mechanosensitive channel of small conductance) is vital for bacterial survival under hypoosmotic stress.
- MscS possesses a transmembrane domain forming an ion pore and a cytoplasmic vestibule that undergoes conformational changes during gating.
Purpose of the Study:
- To investigate the interplay between transmembrane and cytoplasmic residues in MscS during channel gating.
- To determine the role of specific charged residues in MscS mechanosensitivity and inactivation.
Main Methods:
- Site-directed mutagenesis was employed to alter specific amino acid residues within the MscS channel.
- Functional properties, including gating threshold and inactivation rates, were assessed following these mutations.
Main Results:
- Mutating Asp-62 to neutral or basic residues increased the gating threshold and inactivation rate.
- Altering the charge of Arg-128 or Arg-131, located near Asp-62, produced similar effects on MscS gating.
- Reversing the charge of Arg-131 complemented the effects of Asp-62 to arginine mutation, suggesting an electrostatic interaction.
Conclusions:
- The cytoplasmic domain of MscS influences mechanosensitive gating and inactivation rate.
- Electrostatic interactions between Asp-62 and Arg-131 are critical for MscS channel function and regulation.
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