Maleeva Galyna, Peiretti Franck, Zhorov Boris S, Bregestovski Piotr
INSERM, INS, Institut de Neurosciences des Systèmes, Aix-Marseille UniversityMarseille, France.
Department of Cytology, Bogomoletz Institute of PhysiologyKyiv, Ukraine.
Front Mol Neurosci. 2017 May 16;10:125. doi: 10.3389/fnmol.2017.00125. eCollection 2017.
Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2') increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.
尼氟灭酸（NFA）是非甾体抗炎药中芬那酸盐类的一员。该化合物及其衍生物在全球临床中用于缓解慢性和急性疼痛。NFA也是常用的电压门控氯离子通道阻滞剂。在此，我们提供证据表明NFA是氯离子通透型甘氨酸受体（GlyRs）的有效阻滞剂，具有亚基作用异质性。利用膜片钳记录的全细胞模式和分子建模，我们分析了NFA对CHO细胞中表达的同聚体α1ΔIns、α2B、α3L以及异聚体α1β和α2β甘氨酸受体的作用。NFA以电压依赖性方式抑制甘氨酸诱导的电流，其对α2和α3甘氨酸受体的阻断效力高于对α1甘氨酸受体的阻断效力。伍德胡尔分析表明，NFA分别在距外膜表面0.16和0.65的分数电距离处阻断α1和α2甘氨酸受体。因此，α1甘氨酸受体中的NFA结合位点更靠近膜的外部，而在α2甘氨酸受体中它在孔中更深。α1甘氨酸受体孔内衬TM2螺旋（2'水平）细胞质部分的G254A突变增加了NFA的阻断效力，而β亚基的掺入没有显著影响。希尔图分析表明，α1和α2甘氨酸受体分别优选被两个和一个NFA分子阻断。使用蒙特卡罗能量最小化的分子建模为实验数据提供了结构原理，并提出沿孔有多个相互作用位点，NFA可在这些位点抑制离子渗透。
