Salomon Johanna J, Spahn Stephan, Wang Xiaohui, Füllekrug Joachim, Bertrand Carol A, Mall Marcus A
Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), Member of the German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany;
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Am J Physiol Lung Cell Mol Physiol. 2016 Apr 1;310(7):L593-602. doi: 10.1152/ajplung.00321.2015. Epub 2016 Jan 22.
Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.
最近的研究确定溶质载体家族26成员9（SLC26A9）氯离子通道是囊性纤维化（CF）的修饰因子和潜在治疗靶点。然而，对上皮细胞中SLC26A9调控的了解仍然有限，且缺乏用于生化和功能研究的稳定表达的细胞模型。因此，我们通过逆转录病毒转染构建了稳定表达HA标签SLC26A9的Fisher大鼠甲状腺（FRT）上皮细胞，并使用蛋白质免疫印迹法、免疫定位法、全细胞膜片钳技术以及在尤斯灌流小室中的跨上皮生物电研究对SLC26A9的表达和功能进行了表征。我们证实在转染的FRT（SLC26A9-FRT）细胞中，SLC26A9在mRNA和蛋白质水平均稳定表达。免疫定位和蛋白质免疫印迹法在不同的细胞内区室检测到了SLC26A9，在细胞表面检测到的较少。全细胞膜片钳记录显示，与对照转导的FRT（Control-FRT）细胞相比，SLC26A9-FRT细胞的组成型氯离子电流显著增加（P < 0.01）。类似地，跨上皮测量显示，与Control-FRT细胞单层相比，SLC26A9-FRT细胞单层的基础短路电流显著增加（P < 0.01）。SLC26A9介导的氯离子电流通过环磷酸腺苷（cAMP）依赖性刺激（异丁基甲基黄嘌呤和福斯可林）增加，并被GlyH-101、氟尼缩松、二乙氨基二硫代甲酸钠（DIDS）和5-硝基-2-（3-苯丙基氨基）苯甲酸（NPPB）以及参与上皮渗透压调节的WNK1的RNA干扰敲低所抑制。我们的结果支持，这些稳定表达SLC26A9的新型上皮细胞将成为药理学调控研究的有用模型，包括鉴定SLC26A9氯离子通道的激活剂，这些激活剂可能补偿囊性纤维化跨膜传导调节因子（CFTR）介导的氯离子分泌缺陷，并作为CF患者以及潜在其他黏液阻塞性肺部疾病患者的替代治疗靶点。
