Tweety-homolog ( Ttyh ) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC swell ) in the Brain

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl - and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC ) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC in astrocytes. Gene-silencing of all eliminated hypo-osmotic-solution-induced Cl conductance (I ) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the VRAC in the brain.