Interactions between permeant and blocking anions inside the CFTR chloride channel pore

Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.