Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, NY, 12208, USA.
J Physiol. 2017 Nov 15;595(22):6939-6951. doi: 10.1113/JP275053. Epub 2017 Sep 12.
The volume-regulated anion channel (VRAC) is a swelling-activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners. We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling-activated release of diverse organic osmolytes in rat astrocytes. Efflux of uncharged osmolytes (myo-inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E. Conversely, release of charged osmolytes (d-aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D. Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D-containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules.
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.
体积调节阴离子通道（VRAC）是一种肿胀激活的氯离子通道，对无机阴离子和多种小分子有机物质具有通透性。VRAC 通过 LRRC8 蛋白的异源二聚体形成，其中 LRRC8A 是必需的，而 LRRC8B/C/D/E 则作为可交换的互补伴侣。我们使用 RNAi 方法和放射性示踪剂测定法来探索哪些 LRRC8 异构体有助于大鼠星形胶质细胞中各种有机渗透物的肿胀激活释放。未带电渗透物（肌醇和牛磺酸）的流出被 LRRC8A 或 LRRC8D 的缺失抑制，但 LRRC8C+LRRC8E 的缺失不抑制。相反，带电荷渗透物（天冬氨酸）的释放被 LRRC8A 或 LRRC8C+LRRC8E 的缺失强烈抑制，但 LRRC8D 的下调基本不受影响。我们的研究结果表明，在同一细胞类型中存在多种异源 VRAC：LRRC8A/D 包含的异源二聚体似乎主导不带电荷渗透物的释放，而 LRRC8A/C/E 与 LRRC8D 的额外贡献一起形成了带电荷分子的通道。
体积调节阴离子通道（VRAC）是广泛表达的脊椎动物 Cl /阴离子通道，由属于 LRRC8 家族的蛋白质组成，并通过细胞肿胀激活。在大脑中，VRAC 有助于多种小分子有机物质的生理和病理释放，包括氨基酸神经递质谷氨酸、天冬氨酸和牛磺酸。在本工作中，我们探索了所有五个 LRRC8 家族成员在大鼠原代星形胶质细胞中有机渗透物释放中的作用。使用 RNAi 方法修饰 LRRC8 蛋白的表达，并使用放射性示踪剂测定法在受到低渗介质（渗透压降低 30%）挑战的细胞中定量测定氨基酸通量。与我们之前的工作一致，LRRC8A 的敲低强烈且同等地抑制了放射性标记的 d-[ C]天冬氨酸和 [ H]牛磺酸的释放。在其他 LRRC8 亚基中，LRRC8D 的下调强烈抑制了不带电荷的渗透物 [ H]牛磺酸和肌-[ H]肌醇的释放，而对同时测量的带电荷 d-[ C]天冬氨酸的流出没有重大影响。相比之下，d-[ C]天冬氨酸的释放对 LRRC8C+LRRC8E 的缺失更为敏感，但对 LRRC8D 的下调无影响。最后，siRNA 敲低 LRRC8C+LRRC8D 强烈抑制了所有渗透物的释放。总体而言，我们的研究结果表明，星形胶质细胞中至少存在两种不同的异源 VRAC。LRRC8A/D 包含的通透性途径似乎主导不带电荷渗透物的释放，而替代通道（或通道）由 LRRC8A/C/D/E 组成，负责带电荷分子的损失。
