CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) ANION-BINDING AS A PROBE OF THE PORE

We compared the effects of mutations in transmembrane segments (TMs) T M1, TM5, and TM6 on the conduction and activation properties of the cy stic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby , to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution s tudies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conductio n property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord w ith the notion that anion-water interactions are important determinant s of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mu tations in TM5 and TM6 also dramatically reduced the sensitivity of CF TR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected i f these TMs are intimately involved in the physical process that opens and closes the channel.