A TMEM63B variant with enhanced mechanosensitive channel activity and acquired lipid scramblase function

Area of Science:

• Molecular Biology
• Neuroscience
• Biophysics

Background:

• TMEM63B is a mechanosensitive ion channel linked to severe neurodevelopmental disorders.
• Pathogenic variants V44M and T481N alter TMEM63B function, inducing constitutive phospholipid scramblase activity.
• The hydrophobic neck region is critical for channel gating and permeation.

Purpose of the Study:

• To characterize the I475del TMEM63B variant and elucidate the mechanistic basis of channel-to-scramblase switching.
• To define the role of the hydrophobic gate in TMEM63B channelopathies.
• To develop a stratified molecular model for TMEM63B-associated diseases.

Main Methods:

• Characterization of the I475del variant using electrophysiology and functional assays.
• Site-directed mutagenesis to investigate the role of key residues in gating and transport.
• Analysis of ion and lipid transport under varying osmotic conditions.

Main Results:

• The I475del variant exhibits enhanced mechanosensitivity and basal leak currents, classifying it as a gain-of-function variant.
• I475del, like V44M and T481N, possesses constitutive phospholipid scramblase activity.
• I475del shows potentiated scramblase activity under hypotonic stress, and mutations abolishing gating also abolish ion and lipid transport.

Conclusions:

• Pathogenic TMEM63B variants progressively destabilize the hydrophobic gate, leading to altered ion and lipid transport.
• A stratified molecular model explains TMEM63B channelopathies, involving lipid permeation and force-induced ion/lipid cotransport.
• Findings advance understanding of TMEM63B function, disease mechanisms, and potential therapeutic targets.