Medical College, Jinan University, Guangzhou, China.
J Cell Physiol. 2011 Oct;226(10):2516-26. doi: 10.1002/jcp.22596.
In this study, the activation mechanisms of the background chloride current and the role of the current in maintaining of basal cell volume were investigated in human nasopharyngeal carcinoma CNE-2Z cells. Under isotonic conditions, a background chloride current was recorded by the patch clamp technique. The current presented the properties similar to those of the volume-activated chloride current in the same cell line and was inhibited by chloride channel blockers or by cell shrinkage induced by hypertonic challenges. Extracellular applications of reactive blue 2, a purinergic receptor antagonist, suppressed the background chloride current in a concentration-dependent manner under isotonic conditions. Depletion of extracellular ATP with apyrase or inhibition of ATP release from cells by gadolinium chloride decreased the background current. Extracellular applications of micromolar concentrations of ATP activated a chloride current which was inhibited by chloride channel blockers and hypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride. Transfection of CNE-2Z cells with ClC-3 siRNA knocked down expression of ClC-3 proteins, attenuated the background chloride current and prevented activation of the ATP-induced current. Furthermore, knockdown of ClC-3 expression or exposures of cells to ATP (10 mM), the chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, or reactive blue 2 increased cell volume under isotonic conditions. The results suggest that ClC-3 protein may be a main component of background chloride channels which can be activated under isotonic conditions by autocrine/paracrine ATP through purinergic receptor pathways; the background current is involved in maintenance of basal cell volume.
在这项研究中，我们研究了人鼻咽癌 CNE-2Z 细胞中背景氯离子电流的激活机制及其在维持基础细胞体积中的作用。在等渗条件下，我们使用膜片钳技术记录背景氯离子电流。该电流呈现出与同细胞系中体积激活氯离子电流相似的特性，并且被氯离子通道阻滞剂或高渗挑战引起的细胞收缩所抑制。在等渗条件下，外源性应用嘌呤能受体拮抗剂反应蓝 2 以浓度依赖性方式抑制背景氯离子电流。用 apyrase 耗尽细胞外 ATP 或用氯化钆抑制细胞内 ATP 释放，均可降低背景电流。外源性应用毫摩尔浓度的 ATP 可激活氯离子电流，该电流被氯离子通道阻滞剂和高渗溶液抑制。细胞外 ATP 还可以逆转氯化钆的作用。用 ClC-3 siRNA 转染 CNE-2Z 细胞可敲低 ClC-3 蛋白的表达，减弱背景氯离子电流并阻止 ATP 诱导电流的激活。此外，敲低 ClC-3 表达或用 ATP（10mM）、氯离子通道阻滞剂 5-硝基-2-(3-苯丙基氨基)苯甲酸（NPPB）和他莫昔芬以及反应蓝 2 处理细胞，均可在等渗条件下增加细胞体积。结果表明，ClC-3 蛋白可能是背景氯离子通道的主要组成部分，该通道可通过自分泌/旁分泌 ATP 激活嘌呤能受体途径在等渗条件下激活；背景电流参与维持基础细胞体积。
