MULTIION 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 tra nsmembrane 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- chan nels by binding to a site similar to 40% of the way through the transm embrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl- concentration. Increasing extracellular Cl- concentration reduced intracellular gluconate affin ity, suggesting that a repulsive inter-action occurs between Cl- and g luconate ions within the channel pore, an effect that would require th e 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 fi om the outside. Increasing the intracellular Cl- concentration also reduced t ile affinity of intracellular gluconate block, consistent with competi tion between intracellular Cl- and gluconate ions for a common binding site in the pole. Based on this evidence that CFTR is a multi-ion por e, 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 accur acy, including the dependence of blocker affinity on exter1 nal Cl-(bu t 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 frac tion behavior seen in Cl-/SCN- mixtures.