NON-PORE LINING AMINO-ACID SIDE-CHAINS INFLUENCE ANION SELECTIVITY OFTHE HUMAN CFTR CL- CHANNEL EXPRESSED IN MAMMALIAN-CELL LINES

1. The effects of individually mutating two adjacent threonine residue s in the sixth membrane-spanning region (TMB) of the cystic fibrosis t ransmembrane conductance regulator (CFTR) Cl- channel on permeation pr operties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR. 2. A number of mutations of T338 significantly affected the permeation properties of the channel. Incre ases and decreases in single channel conductance were observed for dif ferent mutants. Anion selectivity was strongly affected, with no two c hannel variants sharing the same selectivity sequence. Several mutatio ns led to strong inward rectification of the macroscopic current-volta ge relationship. The effects of these mutations on permeation properti es were correlated with the size of the aminoacid side chain substitut ed, rather than its chemical nature. 3. Most mutations of T339 resulte d in a lack of functional channel expression and apparent misprocessin g of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effec ts were not as strong as for T338 mutations. 4. These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl- channel. It is suggested that mutation of this residue a lters the interaction between permeating anions and the channel pore v ia an indirect effect on the orientation of the TM6 helix.