Billet Arnaud, Froux Lionel, Hanrahan John W, Becq Frederic
Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers - ERL7368, Centre National de la Recherche ScientifiquePoitiers, France.
Department of Physiology, McGill University, MontrealQC, Canada.
Front Pharmacol. 2017 Apr 7;8:195. doi: 10.3389/fphar.2017.00195. eCollection 2017.
The chloride (Cl) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic / relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.
氯离子（Cl）通道囊性纤维化跨膜传导调节因子（CFTR）在囊性纤维化（CF）中存在缺陷，其编码基因突变会导致多种缺陷，如错误折叠的蛋白质在内质网中滞留、质膜稳定性降低、通道门控异常且开放概率低以及热不稳定，进而导致通道在生理温度下失活。药物治疗是CF领域的一种主要治疗方法，需要合理且快速的工具来鉴定有前景的化合物。现有的高通量筛选测定法通常是快速且灵敏的技术，但缺乏特异性。很少有研究使用自动膜片钳（APC）记录CFTR，且没有研究比较传统技术和平面技术，并展示它们在不同类型实验中的能力。在本研究中，我们评估了平面平行APC技术在CFTR转运校正剂和CFTR功能调节剂药理研究中的应用。在优化条件下，我们使用自动全细胞膜片钳记录野生型和校正后的F508del-CFTR Cl电流，并将数据与传统手动全细胞膜片钳获得的结果进行比较。我们发现自动膜片钳和手动膜片钳在膜片钳参数（如细胞电容和串联电阻）上没有显著差异。此外，结果表明两种方法在CFTR电流记录方面具有良好的相似性。我们表明，手动和自动技术都可以使用相似的刺激方案，从而精确控制温度、经典/关系，并及时监测电流稳定性。总之，平行膜片钳记录允许通过各种可用测试快速有效地研究CFTR电流，可被视为CF药物治疗中通量筛选的新工具。
