Multi-Ion Mechanism for Ion Permeation and Block in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

The mechanism of Cl(−) ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl(−) channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl(−) concentration. Increasing extracellular Cl(−) concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl(−) and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl(−) is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl(−) concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl(−) and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl(−) permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl(−) (but not gluconate) ions and the dependence of channel conductance on Cl(−) concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN(−)) ions and anomalous mole fraction behavior seen in Cl(−)/SCN(−) mixtures.