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

Abstract

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]aspartate and [ H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [ H]taurine and myo-[ H]inositol, without major impact on the simultaneously measured efflux of the charged d-[ C]aspartate. In contrast, the release of d-[ C]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.

Cell Volume Control in Healthy Brain and Neuropathologies

“…The functional role for the LRRC8E subunit is particularly interesting and unexpected. In our hands, astrocytes have at least ten-fold lower expression of the LRRC8E mRNA as compared to other LRRC8 transcripts (Hyzinski-Garcia et al, 2014; Schober et al, 2017). Similarly, transcriptomics assays reveal extremely low LRRC8E expression levels in astrocyte and other brain cells (Zhang et al, 2014).…”

“…These may provide additional minor contributions to RVD (Parkerson & Sontheimer, 2004; Liu et al, 2006). The molecular nature of astrocytic VRAC has been uncovered only recently (Hyzinski-Garcia, Rudkouskaya, & Mongin, 2014; Schober, Wilson, & Mongin, 2017) and is further discussed in section 12.8.…”

“…12.2C, D). Interestingly, our recent molecular biology work strongly suggests that there are at least two different heteromeric VRAC complexes, which have preferential selectivity for uncharged and charged organic osmolytes [see (Schober et al, 2017) and additional discussion in section 12.8]. …”

“…These findings led to the conclusion that LRRC8A/LRRC8D-containing heteromers are the long-sought after swelling-activated taurine channel. Several subsequent publications explored the requirements of LRRC8 composition for the release of taurine and other structurally diverse organic osmolytes, including glutamate, GABA glycine, lactate, and myo -inositol (Gaitan-Penas et al, 2016; Lutter, Ullrich, Lueck, Kempa, & Jentsch, 2017; Schober et al, 2017). The emerging picture is interesting but rather complex.…”

“…Our work explored the requirements of endogenous LRRC8 subunits for organic osmolyte release in brain astrocytes (Schober et al, 2017). The principal observations of this study are presented in Fig.…”