Role of LRRC8 Anion Channels in Brain Taurine Signaling and Body Fluid Homeostasis

SUMMARY: Volume-regulated anion channels (VRACs) are ubiquitous chloride/anion channels formed by five proteins from the leucine-rich repeat-containing family 8 (LRRC8A-E), and found in essentially all types of vertebrate cells. These channels are activated by cell swelling and enable cell volume control through the release of cytosolic chloride and a variety of small organic molecules (osmolytes). The brain has been viewed as the place with the highest physiological and pathological relevance for VRACs. Even minor changes in neural cell size have a profound impact on the geometry of extracellular space, ionic balances, and neurotransmitter signaling, thus emphasizing the importance of VRACs and cell volume control. However, such control comes at a steep price: opening of VRACs releases the excitatory neurotransmitters glutamate and aspartate, which drive excitotoxicity in stroke, traumatic brain injury, and epilepsy. For this reason, inhibition of VRAC-induced excita- bility is pursued as a neuroprotective strategy. Yet, such thinking may be overly simplistic because it does not consider VRACs’ contributions to the release of inhibitory molecules. Mice with conditional brain deletion of the essential VRAC subunit LRRC8A (Nestin-Cre driven bLRRC8A KO) die from spontaneous seizures during adolescence and early adulthood. The molecular mechanisms responsible for this lethal phenotype remain an enigma. The current project will address the critical, but previously unknown, role of VRACs in control of brain excitability. Our overarching hypothesis is that LRRC8/VRAC CHANNEL FUNCTION IN GLIAL AND NEURONAL CELLS IS RESPONSIBLE FOR TONIC AND STIMULATED RELEASE OF INHIBITORY NEUROTRANSMITTERS AND NEUROMODULATORS. Such release of inhibitory molecules provides control of brain excitation, and its abrogation may be responsible for lethal seizures in bLRRC8A KO mice. The current exploratory project will address our main hypothesis using two working models. Work in SPECIFIC AIM 1 will conclusively prove the contribution of LRRC8A-containing VRACs to inhibitory control through their regulation of extracellular taurine and GABA levels in mammalian brain. This will be done using a microdialysis approach in the already validated bLRRC8A KO mice. Studies in AIM 2 will ascertain the functional role for astrocytic VRAC-mediated taurine release in hypothalamic regulation of body fluid-electrolyte homeostasis. This will be done by measuring changes in blood osmolarity and activity/c-fos immunoreactivity of hypothalamic magnocellular neurons in mice carrying astrocytic deletion of LRRC8A (Aldh1l1-CreERT2-driven aLRRC8A KO). Overall, this exploratory study will address a substantial gap in knowledge: the role of brain LRRC8/VRAC channels in the negative control of neuronal excitability. This new information is very important for the rational design of neuroprotective agents that target VRAC activity in stroke and other brain pathologies.