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

TMEM63B, a mechanosensitive ion channel (MSC), is associated with severe neurodevelopmental disorders, including severe early-onset developmental and epileptic encephalopathy. Structure-function studies have shown that TMEM63B pathogenic variants, including V44M in transmembrane helix 0 (TM0) and T481N in TM4, cluster near the hydrophobic neck region of the ion permeation pathway, a region critical for gating and permeation. Notably, V44M and T481N convert TMEM63B into constitutive phospholipid scramblases without obvious effects on their MSC activity, revealing an unexpected channel-to-scramblase switch in these variants. To further define the mechanistic basis of this phenomenon, here we characterized I475del, a TM4 deletion variant near V44M and T481N. Unlike V44M and T481N, the I475del channel exhibited basal leak currents, enhanced mechanically activated currents, and elevated mechanosensitivity, altogether supporting its classification as a bona fide gain-of-function MSC variant. Like V44M and T481N, I475del also enabled constitutive phospholipid scramblase activity. However, unlike these variants, I475del uniquely displayed further potentiation of scramblase activity under hypotonic osmotic stress. In addition, mutating the key residues that control gating conformational changes abolished gain-of-function ion and lipid transport through I475del. Together, our results support a stratified molecular model of TMEM63B channelopathies in which the pathogenic variants progressively destabilize the hydrophobic gate, permitting lipid permeation at the resting state followed by force-induced ion and lipid cotransport. These findings advance the mechanistic understanding of TMEM63B function and TMEM63B-associated disease and provide a framework for developing therapeutic strategies targeting variant-specific pathologies.