Abstract
<jats:title>Abstract</jats:title> <jats:p> Piezo1 is a mechanically activated cation channel whose N-linked glycans support protein maturation and plasma membrane trafficking, but their contribution to mechanical gating is unknown. We asked whether hypoglycosylation alters Piezo1 mechanosensitivity and cortical neuronal mechanotransduction, with potential relevance to neurological manifestations of congenital disorders of glycosylation (CDG). Human Piezo1 was studied in HEK293 cells after mutation of two conserved cap-domain N-glycosylation sites or inhibition of N-glycan maturation with swainsonine or kifunensine. Mechanically activated currents were recorded by cell-attached patch-clamp during incremental negative-pressure pulses, whereas Ca <jats:sup>2+</jats:sup> responses were measured during uniaxial stretch. Piezo1 abundance, synaptic localisation and stretch-evoked Ca <jats:sup>2+</jats:sup> signals were also examined in primary mouse cortical neurons. On poly-L-lysine, N2293Q or N2330Q shifted the pressure-response relationship towards lower activating pressures without changing maximal current or inactivation kinetics. This effect was absent on collagen. Swainsonine and kifunensine reduced mature Piezo1 glycosylation and lowered the mechanical activation threshold. Hypoglycosylation enhanced Ca <jats:sup>2+</jats:sup> entry during submaximal stretch in HEK293 cells. In cortical neurons, inhibition of glycan maturation increased somatic Piezo1 immunoreactivity without changing its association with synaptic markers, and potentiated Ca <jats:sup>2+</jats:sup> responses to both the Piezo1 activator Yoda1 and submaximal stretch. Thus, mature N-glycans and the extracellular adhesive environment jointly set Piezo1’s mechanical activation threshold rather than merely regulating biosynthesis and trafficking. These findings establish glycosylation-mechanics coupling as a determinant of neuronal force sensing and suggest that, by facilitating Piezo1 recruitment, defective glycosylation may increase cortical vulnerability to mechanical stress, potentially contributing to head trauma-triggered neurological episodes in phosphomannomutase 2 deficiency (PMM2-CDG). </jats:p> <jats:sec> <jats:title>Key points</jats:title> <jats:list list-type="bullet"> <jats:list-item> <jats:p>Piezo1 channels convert mechanical forces into electrical and calcium signals. N-linked glycans support channel trafficking to the plasma membrane, but whether they tune the force needed for Piezo1 activation was unknown.</jats:p> </jats:list-item> <jats:list-item> <jats:p>Mutating either of two conserved N-glycosylation sites in Piezo1 cap domain, or pharmacologically restricting N-glycan maturation, lowered channel’s mechanical activation threshold without changing maximal current or inactivation.</jats:p> </jats:list-item> <jats:list-item> <jats:p>This sensitisation depended on the adhesive substrate (occurred on poly-L-lysine but not collagen), and was most evident during submaximal stretch, showing that glycosylation and the extracellular mechanical environment jointly determine Piezo1 force sensing.</jats:p> </jats:list-item> <jats:list-item> <jats:p> In mouse cortical neurons, impaired N-glycan maturation increased somatic Piezo1 abundance and enhanced Ca <jats:sup>2+</jats:sup> responses to its chemical activator Yoda1 and stretch, without changing synaptic localisation. </jats:p> </jats:list-item> <jats:list-item> <jats:p>By allowing weak mechanical inputs to recruit Piezo1 more effectively, defective glycosylation may increase cortical responses to mechanical stress and help explain susceptibility to head trauma-triggered neurological episodes in phosphomannomutase 2 deficiency (PMM2-CDG).</jats:p> </jats:list-item> </jats:list> </jats:sec>