CHANNEL-LINING RESIDUES IN THE M3 MEMBRANE-SPANNING SEGMENT OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride-selective channel. Residues from the 12 putative membrane-sp anning segments form at least part of the channel lining. We need to i dentify the channel-lining residues in order to understand the structu ral basis for the channel's functional properties. Using the substitut ed-cysteine-accessibility method we mutated to cysteine, one at a time , 24 consecutive residues (Asp192-Ile215) in the M3 membrane-spanning segment. Cysteines substituted for His199, Phe200, Trp202, Ile203, Pro 205, Gln207, Leu211, and Leu214 reacted with charged, sulfhydryl-speci fic reagents that are derivatives of methanethiosulfonate (MTS). We in fer that these residues are on the water-accessible surface of the pro tein and probably form a portion of the channel Lining. When plotted o n an alpha-helical wheel the exposed residues from Gln207 to Leu214 li e within an are of 60 degrees; the exposed residues in the cytoplasmic half (His199-Ile203) lie within an are of 160 degrees. We infer that the secondary structures of the extracellular and cytoplasmic halves o f M3 are alpha-helical and that Pro205, in the middle of the M3 segmen t, may bend the M3 segment, moving the cytoplasmic end of the segment in toward the central axis of the channel. The bend in the M3 segment may help to narrow the channel lumen near the cytoplasmic end. In addi tion, unlike full-length CFTR, the current induced by the deletion con struct, Delta 259, is inhibited by the MTS reagents, implying that the channel structure of Delta 259 is different than the channel structur e of wild-type CFTR.