Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties

Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO₂)₄^2- ions to weaken the pore-blocking effect of intracellular Pt(NO₂) ₄^2- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO₂)₄^2- ions. These mutations do not affect unitary Cl^- conductance or block of single-channel currents by extracellular Pt(NO₂)₄^2- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.