Friard Jonas, Tauc Michel, Cougnon Marc, Compan Vincent, Duranton Christophe, Rubera Isabelle
LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France.
Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de MontpellierMontpellier, France.
Front Pharmacol. 2017 May 31;8:328. doi: 10.3389/fphar.2017.00328. eCollection 2017.
Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16A-A01, a so-called specific inhibitor of calcium-activated Cl conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTR-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTR-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.
氯离子通道在多种生理功能及人类疾病中发挥着重要作用。从历史上看，由于缺乏强大的选择性药理试剂来克服表征这些通道分子身份的技术挑战，氯离子通道领域长期以来一直被忽视。最近，LRRC8家族成员已被证明对于产生容积调节性阴离子通道（VRAC）电流至关重要，VRAC电流是一种氯离子电导，控制着调节性容积减小（RVD）过程。在表达LRRC8蛋白且缺乏其他类型氯离子通道（CFTR和ANO1/2）的野生型HEK-293细胞中，测试了六种常用氯离子通道抑制剂对VRAC/LRRC8介导的氯离子转运的抑制作用。我们使用膜片钳全细胞方法以及基于荧光的低渗刺激期间细胞容积变化定量来探究抑制剂的有效性。DCPIB和NFA在全细胞配置中均抑制VRAC电流，其IC值分别为5±1μM和55±2μM。令人惊讶的是，两种CFTR抑制剂GlyH - 101和PPQ - 102在其当前使用浓度范围内也抑制VRAC电导，IC值分别为10±1μM和20±1μM。T16A - A01是一种所谓的钙激活氯离子电导特异性抑制剂，可以阻断低渗刺激引发的氯离子电流，IC值为6±1μM。此外，这些抑制剂显著降低了低渗刺激后的RVD。CFTR - 172是唯一对VRAC/LRRC8介导的氯离子电导几乎没有影响的抑制剂。除CFTR - 172外，所有测试的抑制剂均抑制VRAC/LRRC8介导的氯离子电导以及低渗刺激期间的细胞容积变化。这些结果揭示了氯离子通道抑制剂明显缺乏特异性的问题，并提出了这些抑制剂实际上如何阻断氯离子电导的疑问。
