Interactions between Impermeant Blocking Ions in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore: Evidence for Anion-Induced Conformational Changes

Abstract

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(NO(2))(4) (2-) ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO(2))(4) (2-), glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO(2))(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(NO(2))(4) (2-) ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO(2))(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.