Probing CFTR Channel Structure and Function Using the Substituted-Cysteine-Accessibility Method

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel whose activation is regulated by phosphorylation and by ATP binding and hydrolysis (1 -4 ). The functional properties of the channel have been extensively studied using electrophysiological techniques (4 ). Less is known about the structural bases for the functional properties. The cloning of CFTR in 1989 provided the primary amino acid sequence and a putative transmembrane topology of the protein (5 ). In order to understand the structural bases for the functional properties of the channel, we sought to identify the residues lining the ion channel because they are likely to be the major determinants of the channel’s functional properties. Although the channel-lining residues lie within membrane-spanning segments, they are part of the water-accessible surface of the protein. The substituted-cysteine-accessibility method (SCAM), which we developed, provides an approach to identify systematically the channel-lining residues (6 -9 ). We have applied SCAM to three of CFTR’s 12 putative membrane-spanning segments (9 -11 ).