Mutation-induced Blocker Permeability and Multiion Block of the CFTR Chloride Channel Pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO₂)₄^2- acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent K _d 556 μM), kinetically fast, and weakened by extracellular Cl ^- ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO₂)₄^2- ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO₂) ₄^2- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO₂)₄^2- ions to the extracellular solution. This "punchthrough" was prevented by extracellular Cl^- ions, reminiscent of a "lock-in" effect. Relief of block in F337A by Pt(NO₂)₄^2- permeation was only observed for blocker concentrations above 300 μM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl^- current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO₂)₄ ^2- in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO ₂)₄^2- to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO₂)₄^2- ions within the pore promotes their permeation to the extracellular solution.