Molecular composition and heterogeneity of the LRRC8-containing swelling-activated osmolyte channels in primary rat astrocytes

The volume-regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl-/ anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled D-[C-14] aspartate and [H-3] taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [H-3] taurine and myo-[H-3] inositol, without major impact on the simultaneouslymeasured efflux of the charged D-[C-14] aspartate. In contrast, the release of D-[C-14] aspartate was preferentially sensitive to deletion of LRRC8C+ LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+ LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D-containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules.