IDENTIFICATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATORCHANNEL-LINING RESIDUES IN AND FLANKING THE M6 MEMBRANE-SPANNING SEGMENT

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding . Work by others suggested that some residues in the sixth transmembra ne segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are e xposed in the channel and the secondary structure of M6, we used the s ubstituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessib ility of the engineered cysteines to charged, lipophobic, sulfhydryl-s pecific methanethiosulfonate (MTS) reagents applied extracellularly. T he cysteines substituted for lle331, Leu333, Arg334, Lys335, Phe337, S er341, lle344, Arg347, Thr351, Arg352, and Gln353 reacted with the MIS reagents, and we infer that they are exposed on the water-accessible surface of the protein, From the pattern of the exposed residues we in fer that the secondary structure of the M6 segment includes both alpha -helical and extended regions. The diameter of the channel from the ex tracellular end to the level of Gln353 must be at least 6 Angstrom to allow the MTS reagents to reach these residues.