Received for publication October 30, 2006.
Revised February 8, 2007.
Accepted for publication February 8, 2007.

Identification of a Second Blocker Binding Site at the Cytoplasmic Mouth of the CFTR Chloride Channel Pore

Abstract

Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel is inhibited by a broad range of substances that bind within a wide inner vestibule in the pore and physically occlude Cl^- permeation. Binding of many of these so-called open channel blockers involves electrostatic interactions with a positively charged lysine residue (K95) located in the pore. Here we use site-directed mutagenesis to identify a second blocker binding site located at the cytoplasmic mouth of the pore. Mutagenesis of a positively charged arginine at the cytoplasmic mouth of the pore, R303, leads to significant weakening of the blocking effects of suramin, a large negatively charged organic molecule. Apparent suramin affinity is correlated with the side chain charge at this position, consistent with an electrostatic interaction. In contrast, block by suramin is unaffected by mutagenesis of K95, suggesting that it does not approach close to this important pore-forming lysine residue. We propose that the CFTR pore inner vestibule contains two distinct blocker binding sites. Relatively small organic anions enter deeply into the pore to interact with K95, causing an open channel block that is sensitive to both the membrane potential and the extracellular Cl^- concentration. Larger anionic molecules can become lodged in the cytoplasmic mouth of the pore where they interact with R303, causing a distinct type of open channel block that is insensitive to membrane potential or extracellular Cl^- ions. The pore may narrow significantly between the locations of these two blocker binding sites.

Key words: Chloride, Ion transporters (SERCA, Na/K ATPase, CFTR), Func. analysis receptor/ion channel mutants