CONTRIBUTION OF PROLINE RESIDUES IN THE MEMBRANE-SPANNING DOMAINS OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR TO CHLORIDE CHANNEL FUNCTION

Proline residues located in membrane spanning domains of transport pro teins are thought to play an important structural role, In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted tra nsmembrane segments contain four prolines: Pro(99), Pro(205), Pro(324) , and Pro(1021). These residues are conserved across species, and muta tions of two (P99L and P205S) are associated with cystic fibrosis, To evaluate the contribution of these prolines to CFTR Cl- channel functi on, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A), We also cons tructed the two cystic fibrosis-associated mutations, cAMP agonists st imulated whole cell Cl- currents in HeLa cells expressing the individu al constructs that resembled those produced by wild-type CFTR, However , the amount of current was decreased in the rank order: wild-type CFT R = Pro(324) > Pro(1021) > Pro(99) greater than or equal to Pro(205) m utants, The anion selectivity sequence of the mutants (Br- greater tha n or equal to Cl- > I-) resembled wild-type except for P99L (Br- great er than or equal to Cl- = I-). Although the Pro(99), Pro(324) , and Pr o(1021) mutants produced mature protein, the amount of mature protein was much reduced with the pro205 mutants, and the P-Quad-A made none. Because the Pro(99) constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties, Mutant channels were regulated like wild-type CFTR; however, single-c hannel conductance was decreased in the rank order: wild-type CFTR gre ater than or equal to P99G > P99L greater than or equal to P99A. These results suggest that proline residues in the transmembrane segments a re important for CFTR function, Pro(205) is critical for correct prote in processing, and Pro(99) may contribute either directly or indirectl y to the Cl- channel pore.