Interactions between impermeant blocking ions in the cystic fibrosis transmembrane conductance regulator chloride channel pore: Evidence for anion-induced conformational changes

It is well known that extracellular Cl^- ions can weaken the inhibitory effects of intracellular open channel blockers in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel pore. This effect is frequently attributed to repulsive ion-ion interactions inside the pore. However, since Cl^- ions are permeant in CFTR, it is also possible that extracellular Cl^- ions are directly competing with intracellular blocking ions for a common binding site; thus, this does not provide direct evidence for multiple, independent anion binding sites in the pore. To test for the possible through-space nature of ion-ion interactions inside the CFTR pore, we investigated the interaction between impermeant anions applied to either end of the pore. We found that inclusion of low concentrations of impermeant Pt(NO₂) ₄ ^2- ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO₂) ₄ ^2- , glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO₂) ₄ ^2- ions are too strong to be accounted for by simple competitive models of ion binding inside the pore. In addition, extracellular Fe(CN) ₆ ^3- ions, which do not appear to enter the pore, also weaken the blocking effects of intracellular Pt(NO₂) ₄ ^2- ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO₂) ₄ ^2- and Fe(CN) ₆ ^3- binding to an extracellularly accessible site outside of the channel permeation pathway alters the structure of an intracellular anion binding site, leading to weakened binding of intracellular blocking ions.