Excitotoxic neuronal death requires superoxide entry into neurons through volume-regulated anion channels

Neuronal death in stroke and other conditions stems in part from stimulation of <i>N</i>-methyl-d-aspartate (NMDA)-type glutamate receptors. This induces neuronal production of both nitric oxide and superoxide, which together induce oxidative cell injury. Nitric oxide can readily cross lipid membranes, but superoxide, being an anion, cannot. Using primary neuronal cultures, we show that superoxide enters neurons through volume-regulated anion channels (VRACs). Oxidative injury produced by either exogenous superoxide or NMDA receptor stimulation is prevented by the VRAC inhibitor [4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl)oxybutyric acid] (DCPIB) and by disruption of the essential VRAC subunit, leucine-rich repeat-containing 8A (LRRC8A). In mouse cortex, neuronal oxidative injury induced by either NMDA or transient ischemia is likewise blocked by both DCPIB and LRRC8A disruption. Selective expression of LRRC8A/C and LRRC8A/D but not LRRC8A/D subunits conferred superoxide conductance in HeLa cells. Superoxide entry through VRACs is thus a requisite step in excitotoxic neuronal injury, and interventions affecting VRAC subunit composition, localization, or opening could influence neuronal survival.