Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR - Evidence for disruption of a salt bridge

Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmemb rane conductance regulator (CFTR) is a site of four cystic fibrosis-associa ted mutations. To better understand the function of Arg-347 and to learn ho w mutations at this site disrupt channel activity, we mutated Arg-347 to As p, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every A rg-347 mutation examined, except R347K, had a destabilizing effect on the p ore, causing the channel to flutter between two conductance states. Chlorid e flow through the larger conductance state was similar to that of wildtype CFTR, suggesting that the residue at position 347 does not interact direct ly with permeating anions, We hypothesized that Arg-347 stabilizes the chan nel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutat ions. Interestingly, the D924R mutation complemented R347D, yielding a chan nel that behaved Like wild-type CFTR, These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt b ridge with Asp-924; cystic fibrosis-associated mutations disrupt this inter action.