11] Patch-clamp studies of cystic fibrosis transmembrane conductance regulator chloride channel

Abstract

Publisher Summary The cystic fibrosis transmembrane conductance regulator (CFTR) has two sets of six membrane-spanning regions (TM1-TM12), two nucleotidebinding folds (NBF1, NBF2), and a regulatory domain containing numerous potential sites for phosphorylation by protein kinases. It belongs to an important superfamily of ATP-binding cassette (ABC) transport proteins that has members in bacteria, yeast, and higher eukaryotes. Although it may have multiple functions, it is generally accepted that CFTR functions as an ATP-dependent, phosphorylation-activated CI channel. This chapter describes the methods that have been found useful for studying the channel activity of CFTR. Functional studies of endogenous CFTR have been carried out using epithelial and cardiac cells. However, because it is often difficult to obtain giga-ohm seals on native epithelial cells, heterologous expression systems are generally preferred for biophysical studies. Many cell lines have been used successfully for transient and stable transfection, including Chinese hamster ovary (CHO) cells, fibroblasts, baby hamster kidney cells (BHK), and human enbryonic kidney (HEK 293) cells. The choice of preparations depends on the purpose of the experiment.