Kostritskaia Yulia, Pervaiz Sumaira, Klemmer Anna, Klüssendorf Malte, Stauber Tobias
Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany.
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
J Physiol. 2024 Nov 4. doi: 10.1113/JP286665.
Volume-regulated anion channels (VRACs) formed by leucin-rich repeat containing 8 (LRRC8) proteins play a pivotal role in regulatory volume decrease by mediating the release of chloride and organic osmolytes. Apart from the regulation of cell volume, LRRC8/VRAC function underlies numerous physiological processes in vertebrate cells including membrane potential regulation, glutamate release and apoptosis. LRRC8/VRACs are also permeable to antibiotics and anti-cancer drugs, representing therefore important therapeutic targets. The activation mechanisms for LRRC8/VRACs are still unclear. Besides through osmotic cell swelling, LRRC8/VRACs can be activated by various stimuli under isovolumetric conditions. Sphingosine-1-phosphate (S1P), an important signalling lipid, which signals through a family of G protein-coupled receptors (GPCRs), has been reported to activate LRRC8/VRACs in several cell lines. Here, we measured inter-subunit Förster resonance energy transfer (FRET) and used whole-cell patch clamp electrophysiology to investigate S1P-induced LRRC8/VRAC activation. We systematically assessed the involvement of GPCRs and G protein-mediated signal transduction in channel activation. We found that S1P-induced channel activation is mediated by S1PR1 in HeLa cells. Following the downstream signalling pathway of S1PR1 and using toxin-mediated inhibition of the associated G proteins, we showed that Gβγ dimers rather than Gαi or Gαq play a critical role in S1P-induced VRAC activation. We could also show that S1P causes protein kinase D (PKD) phosphorylation, suggesting that Gβγ recruits phospholipase Cβ (PLCβ) with the consequent PKD activation by diacylglycerol. Notably, S1P did not activate LRRC8/VRAC in HEK293 cells, but overexpression of Gβγ-responsive PLCβ isoform could facilitate S1P-induced LRRC8/VRAC currents. We thus identified S1PR1-mediated Gβγ-PLCβ signalling as a key mechanism underlying isosmotic LRRC8/VRAC activation. KEY POINTS: Leucin-rich repeat containing 8 (LRRC8) anion/osmolyte channels are involved in multiple physiological processes where they can be activated as volume-regulated anion channels (VRACs) by osmotic cell swelling or isovolumetric stimuli such as sphingosine-1-phosphate (S1P). In the present study, using pharmacological modulation and gene-depleted cells in patch clamp recording and optical monitoring of LRRC8 activity, we find that LRRC8/VRAC activation by S1P is mediated by the G protein-coupled receptor S1PR1 coupled to G proteins of the Gi family. The signal transduction to LRRC8/VRAC activation specifically involves phospholipase Cβ activation by βγ subunits of pertussis toxin-insensitive heteromeric Gi proteins. S1P-mediated and hypotonicity-induced LRRC8/VRAC activation pathways converge in protein kinase D activation.
由富含亮氨酸重复序列8（LRRC8）蛋白形成的容积调节性阴离子通道（VRAC）通过介导氯离子和有机渗透溶质的释放，在调节性容积减小过程中起关键作用。除了调节细胞容积外，LRRC8/VRAC功能是脊椎动物细胞中众多生理过程的基础，包括膜电位调节、谷氨酸释放和细胞凋亡。LRRC8/VRACs对抗生素和抗癌药物也具有通透性，因此是重要的治疗靶点。LRRC8/VRACs的激活机制仍不清楚。除了通过渗透性细胞肿胀激活外，LRRC8/VRACs还可在等容条件下被各种刺激激活。鞘氨醇-1-磷酸（S1P）是一种重要的信号脂质，通过G蛋白偶联受体（GPCR）家族进行信号传导，据报道它可在几种细胞系中激活LRRC8/VRACs。在此，我们测量了亚基间荧光共振能量转移（FRET），并使用全细胞膜片钳电生理学来研究S1P诱导的LRRC8/VRAC激活。我们系统地评估了GPCRs和G蛋白介导的信号转导在通道激活中的作用。我们发现S1P诱导的通道激活在HeLa细胞中由S1PR1介导。沿着S1PR1的下游信号通路并使用毒素介导的对相关G蛋白的抑制，我们表明Gβγ二聚体而非Gαi或Gαq在S1P诱导的VRAC激活中起关键作用。我们还可以表明S1P导致蛋白激酶D（PKD）磷酸化，这表明Gβγ募集磷脂酶Cβ（PLCβ），随后二酰甘油激活PKD。值得注意的是，S1P在HEK293细胞中未激活LRRC8/VRAC，但Gβγ反应性PLCβ同工型的过表达可促进S1P诱导的LRRC8/VRAC电流。因此，我们确定S1PR1介导的Gβγ-PLCβ信号传导是等渗LRRC8/VRAC激活的关键机制。要点：富含亮氨酸重复序列8（LRRC8）阴离子/渗透溶质通道参与多种生理过程，在这些过程中它们可通过渗透性细胞肿胀或等容刺激（如鞘氨醇-1-磷酸（S1P））被激活为容积调节性阴离子通道（VRAC）。在本研究中，我们在膜片钳记录和LRRC活性的光学监测中使用药理学调节和基因缺失细胞，发现S1P对LRRC8/VRAC的激活由与Gi家族G蛋白偶联的G蛋白偶联受体S1PR1介导。向LRRC8/VRAC激活的信号转导具体涉及百日咳毒素不敏感异源三聚体Gi蛋白的βγ亚基对磷脂酶Cβ的激活。S1P介导的和低渗诱导的LRRC8/VRAC激活途径在蛋白激酶D激活中汇聚。
