TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels.

Area of Science:

• Molecular and Cellular Biology
• Biophysics
• Structural Biology

Background:

• OSCA and TMEM63 proteins are known mechanically activated (MA) ion channels involved in cellular mechanotransduction.
• Typically, OSCAs, TMEM16, and TMC proteins form homodimers with dual pores, suggesting oligomerization is crucial for their function.
• The structural basis and functional implications of TMEM63 proteins, particularly their oligomeric state, remain incompletely understood.

Purpose of the Study:

• To investigate the oligomeric state and structural characteristics of TMEM63 proteins (TMEM63A and TMEM63B).
• To determine the functional properties, including conductance and activation thresholds, of TMEM63 ion channels.
• To explore the role of the intracellular linker IL2 and its evolutionary variations in OSCA/TMEM63 channel gating and oligomerization.

Main Methods:

• X-ray crystallography was employed to determine the high-resolution structures of TMEM63A and TMEM63B.
• Electrophysiological recordings (e.g., patch-clamp) were used to analyze the mechanosensitive currents of TMEM63s and engineered OSCA1.2 channels.
• Site-directed mutagenesis was performed to investigate the function of the IL2 linker and specific residues in channel gating and oligomerization.

Main Results:

• TMEM63A and TMEM63B were found to exist in a monomeric configuration, featuring a single, highly restricted ion pore.
• Functional analysis confirmed TMEM63s as bona fide mechanosensitive ion channels with small conductance and high activation thresholds.
• Modifications to the IL2 linker, including replacement with OSCA1.2 IL2 or mutations, resulted in monomeric OSCA1.2 channels and elevated MA current thresholds.

Conclusions:

• TMEM63 proteins represent a unique class of monomeric mechanosensitive ion channels, distinct from dimeric OSCAs.
• The intracellular linker IL2 and its structural variations play a critical role in modulating channel oligomerization and mechanosensitivity thresholds.
• These findings suggest conserved and divergent gating mechanisms across the OSCA/TMEM63, TMEM16, and TMC channel families.