Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System
Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System
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使用平面膜片钳系统表征 CFTR 激活剂和抑制剂
DOI：
10.1016/j.bpj.2016.11.2561
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
N. Fertig
中科院分区：
文献类型：
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作者：
A. Brüggemann;S. Friis;T. Strassmeier;Markus Rapedius;T. Goetze;Ilka Rinke;C. Haarmann;Nina Brinkwirth;Atsushi Ohtsuki;T. Oka;M. George;N. Fertig
囊性纤维化是由氯离子通道囊性纤维化跨膜调节器（CFTR）的功能障碍引起的。CFTR在上皮细胞的顶膜中表达，其中其参与调节穿过上皮的流体运输。已知蛋白质中的大量突变会导致CFTR功能障碍，并且只有少数药物化合物被开发出来通过恢复通道的氯离子电导来治疗该疾病（1）。CFTR由cAMP依赖性磷酸化激活，并由ATP门控。激活通常使用毛喉素实现，毛喉素激活腺苷酸环化酶，然后通过蛋白激酶A（PKA）导致通道的磷酸化。CFTR激活剂的筛选通常在不存在毛喉素的情况下进行，但在低浓度cAMP的存在下进行。在动物模型中的研究支持CFTR抑制剂用于肠毒素介导的肾小球疾病和多囊肾疾病的抗分泌治疗的开发。为此目的，可以使用替代方法。在内部氟化物的存在下，CFTR被激活到与毛喉素激活后相同的程度。我们在这里提供了来自Nanion的SyncroPatch 384 PE的数据，其中我们在无氟条件下用毛喉素激活通道，或者通过内部溶液交换将氟化物冲洗到细胞内溶液中。我们的研究结果表明，无论是福司可林或细胞内F-激活CFTR是敏感的格列本脲和CFTRInh-172的剂量和/或电压依赖性的方式。总之，这些实验显示了以高通量研究CFTR药理学的稳定和成本优化的方法，这可能为CFTR药物发现提供新的方法。
Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion's SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F-is sensitive to glibenclamide and CFTRInh-172 in a dose-and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR.