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Multi-ion block and blocker permeation in a CFTR channel pore mutant



Multi-ion block and blocker permeation in a CFTR channel pore mutant



Biophysical Journal 86(1): 283a



Open channel block of CFTR Cl- channels expressed in mammalian cell lines by impermeant Pt(NO2)42- ions was studied using inside-out patch clamp recording. Block of wild type CFTR by intracellular Pt(NO2)42- was of modest affinity (Kdapprx556muM), voltage dependent, 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 Pt(NO2)42-. 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)42- increased at very negative voltages in F337A but not wild type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42- 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)42- permeation was only observed for blocker concentrations above 300 muM; 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)42- in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42- to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42- ions within the pore promotes their permeation to the extracellular solution.

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Accession: 035349263

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