Department of Internal Medicine.
Abboud Cardiovascular Research Center, University of Iowa, Iowa City, Iowa, USA.
JCI Insight. 2017 Mar 9;2(5):e90632. doi: 10.1172/jci.insight.90632.
The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pH) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived HO The molecular identity of both the hypoosmolarity-induced and acid pH-conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox-mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pH reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate-induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pH in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.
富含亮氨酸重复蛋白 8A（LRRC8A），也称为 SWELL1，是容积调节阴离子通道（VRAC）的必需组成部分，该通道可被细胞肿胀和离子强度激活。据我们所知，这是首次在结状神经节神经元的原代细胞培养物中报告其表达，并在体、神经突和神经元膜中定位。我们表明，这种神经元 VRAC/SWELL1 除了低渗度外，还能感知低细胞外 pH（pH）。在短暂暴露于细胞外酸性 pH 后，77%的分离结状神经元中可见到强大而持续的氯离子电流。其激活涉及质子外排、细胞内碱化和 NOX 衍生的 HO 的增加。低渗诱导和酸 pH 条件下 VRAC 的分子特征均被 Cre-flox 介导的 KO、shRNA 介导的敲低以及 HEK 细胞和原代结状神经元培养物中的 CRISPR/Cas9 介导的 LRRC8A 缺失所证实。VRAC 的低 pH 激活可减少模拟缺血和 N-甲基-D-天冬氨酸诱导（NMDA 诱导）凋亡期间的神经元损伤。这些结果确定了 VRAC（LRRC8A）作为迷走传入神经元低渗度和低 pH 的双重传感器，并定义了其激活机制及其神经保护潜力。
