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

Abstract

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 swell ) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC swell in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl − conductance (I Cl,swell ) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I Cl,swell with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I Cl,swell 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 bona fide VRAC swell in the brain.

The molecular mechanism of synaptic activity‐induced astrocytic volume transient

“…Likewise, VRAC has long been proposed to be responsible for the transient volume decrease. We have recently reported that astrocytic TTYH1/2/3 are necessary and sufficient for the regulated volume decrease (RVD) in the hippocampus rather than the transient volume decrease (Han et al 2019). RVD is induced by low-frequency stimulation (1 Hz) for 30 min, which is clearly distinguishable from the transient volume decrease, which is induced by a brief train of high frequency stimulations.…”

“…We next tested a possible candidate member of the VRAC Tweety-homolog (TTYH) family, which we recently reported to encode the pore-forming subunits of the swelling-dependent volume-regulated anion channel (VRAC swell ) in the brain (Han et al 2019), as a target for transient volume decrease. We injected a mixture of viruses carrying the previously confirmed shRNA for Ttyh1, Ttyh2 and Ttyh3 (Han et al 2019) into hippocampus. We observed no significant difference in IOS baseline change or decay time between Ttyh 1,2,3 triple gene-silenced condition and control shRNA condition (Fig.…”

“…Various potential molecular candidates have been proposed in the past; Kir4.1 (inwardly rectifying K + channel), NKCC1 (Na + , K + , 2Cl − cotransporter), Na + /K + -ATPase, and K2P (two-pore domain K + channels) (MacVicar et al 2002;Su et al 2002;Djukic et al 2007;Pasler et al 2007;Macaulay & Zeuthen, 2012). Likewise, the molecular identity of the anion channel which is responsible for the efflux of Cl − and osmolytes has not been determined, though it has been proposed that the volume regulated anion channel (VRAC) might be involved (Mulligan & MacVicar, 2006;Okada et al 2009;Han et al 2019;Woo et al 2019;Yang et al 2019).…”

Osmolytes: A Possible Therapeutic Molecule for Ameliorating the Neurodegeneration Caused by Protein Misfolding and Aggregation

“…It is significant for the appropriate functionality and well-being of the nervous system and is strongly prohibited by a particular cell type called astrocytes, owing to their high and exclusive expression of the water channel, aquaporin-4 [50,51]. Water-influx through AQP4 is initiated by osmolytes that activate an outflow of Cl-and osmolytes by means of some volume-regulated anion channel, and afterwards there is an outflow of water to re-establish the volume [52][53][54][55].…”

“…Its levels are associated with osmolytes in the visual but not in the sensorimotor region, indicating the regional specificity of modifications in GABAergic tone in hepatic encephalopathy. Glutamate and other osmolytes are expected to be released through receptors and influence synaptic conduction as well as N-methyl-D-aspartate receptor or metabotropic glutamate receptor-dependent synaptic plasticity [13,55,58]. Alzheimer's disease (AD) is considered a permanent brain disorder that gradually demolishes cognitive functions and ultimately a person's capacity to execute everyday life tasks and behavior.…”