Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore

1. Anion binding within the pores of wild-type and mutant cystic fibrosis t ransmembrane conductance regulator (CFTR) Cl- channels, expressed in two di fferent mammalian cell lines, was assayed using patch clamp recording. Spec ifically experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the po re. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the c onductance sequence Cl- > NO3- > Br- greater than or equal to formate > F- > SCN- approximate to ClO4.- 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide cu rrents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentratio ns (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of thes e anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects ar e consistent with alter ed anion binding within the pore. 6. The effects of mutations on anion permeability a,nd relative anion condu ctance suggested that, for most anions, increased permeability was associat ed with increased conductance. This indicates that the CFTR, channel pore d oes not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the reg ion around F337 and T338 facilitates: their passage through the pore. In wi ld-type CFTR channels, anion entry into this crucial pore region is probabl y dominated by anion hydration energies.