Abstract
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 I/V 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.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride (Cl−) channel whose encoding gene is mutated in the common autosomal recessive disease cystic fibrosis (CF; Riordan et al, 1989)
We describe its use for recording currents carried by wt-CFTR and rescued F508delCFTR channels and compare the results with those obtained from manual patch clamp (MPC) recordings
We tested two automated patch clamp (APC) devices: the Patchliner resistance
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride (Cl−) channel whose encoding gene is mutated in the common autosomal recessive disease cystic fibrosis (CF; Riordan et al, 1989). Cystic fibrosis transmembrane conductance regulator pharmacotherapy is one major therapeutic strategy that involves identifying compounds that correct abnormal CFTR protein. Most research effort is directed at finding small molecules which interact directly with mutated CFTR to correct its folding and gating, and improve protein trafficking and channel function. The search for innovative small molecules and understanding their mechanisms of action remain a priority for CF therapeutic research. To this end, robust screening assays are essential for rapidly testing large numbers of compounds and performing informative concentration–response curves in vitro. As multiple CFTR defects need to be corrected, cell-based assays should monitor improvements in the trafficking, membrane stability, and channel function of rescued mutant CFTR
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