2017 - Activation of CFTR channels in absence of internal fluoride using a highly parallel automated patch clamp system

icon sp96   SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, BPS Meeting 2017  logo pdf   (1.5 MB)


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 of the protein are known to cause CFTR to become dysfunctional and only very few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel. CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which leads to phosphorylation of the channel via protein kinase A (PKA). An alternative mode of activation is via application of intracellular F-. However, intracellular F- has also been widely used on most automated patch clamp (APC) platforms due to it’s stabilizing effects during the giga-seal formation. On the other hand, this also makes it difficult to control the degree of activation of the channel and represents a general limitation in the use of APC platforms in drug discovery for CFTR. We now here present data from Nanion’s SyncroPatch 384PE where we activate the channel with forskolin under F--free conditions at a high troughput. Further, CFTR could also be activated by first patching the cells in F--free conditions and then exposing the cell to intracellular F- using the internal solution exchange function.
Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to Glibenclamide in a dose – and voltage dependent manner. Taken together, these experiments firstly show a fluoride-free approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR.


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