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.