TWEETY-HOMOLOG (TTYH) FAMILY IS STRETCH-SENSITIVE ANION CHANNEL IN ASTROCYTES

Brain volume regulation is essential for maintaining ionic and osmotic homeostasis required for proper nervous system function. Astrocytes play a central role in this process through diverse ion channels, transporters, and abundant water channels. Under hypo-osmotic conditions, water influx leads to cell swelling accompanied by intracellular ionic dilution. Here, we identify the Tweety homologs (TTYH1, TTYH2, and TTYH3) as pore-forming subunits of mechano-sensitive anion channels that open in response to membrane stretch during swelling, in contrast to LRRC8A-containing volume-regulated anion channels (VRACs), which are activated by ionic dilution. Whole-cell patch-clamp recordings with positive-pressure injection through the patch pipette evoked chloride currents in astrocytes, which were abolished by silencing Ttyh1/2/3. Notably, Arg213, a putative pore-forming residue in TTYH1, was indispensable for pressure-induced Cl⁻ conductance. Cholesterol binding suppresses TTYH channel opening at rest, whereas acute cholesterol depletion with methyl-β-cyclodextrin (MβCD) robustly induces TTYH-mediated Cl⁻ currents, consistent with regulation via a putative cholesterol-binding pocket formed by an extracellular hydrophobic cavity, beneath which Arg213 resides as a critical regulatory residue. Reconstitution of hTTYH1 into cholesterol-free liposomes yielded a near-maximal open probability (~1.0), indicating constitutive channel activity. Together, these findings establish the TTYH family as bona fide mechano-sensitive anion channels essential for astrocyte volume regulation.