Contribution of Proline Residues in the Membrane-spanning Domains of Cystic Fibrosis Transmembrane Conductance Regulator to Chloride Channel Function

• Published in: The Journal of biological chemistry Vol. 271; no. 25; pp. 14995 - 15001
• Main Authors: Sheppard, D N, Travis, S M, Ishihara, H, Welsh, M J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21.06.1996
• Subjects: Adenosine Triphosphate - pharmacology; Amino Acid Sequence; Chloride Channels - physiology; Cyclic AMP - analogs & derivatives; Cyclic AMP - pharmacology; Cyclic AMP - physiology; Cystic Fibrosis - genetics; Cystic Fibrosis - physiopathology; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - physiology; Electrophysiology; HeLa Cells; Humans; Membrane Potentials - drug effects; Models, Structural; Point Mutation; Proline; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; vaccinia virus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.25.14995

Proline residues located in membrane-spanning domains of transport proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro 99 , Pro 205 , Pro 324 , and Pro 1021 . These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. To evaluate the contribution of these prolines to CFTR Cl − channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A). We also constructed the two cystic fibrosis-associated mutations. cAMP agonists stimulated whole cell Cl − currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR. However, the amount of current was decreased in the rank order: wild-type CFTR = Pro 324 > Pro 1021 > Pro 99 ≥ Pro 205 mutants. The anion selectivity sequence of the mutants (Br − ≥ Cl − > I − ) resembled wild-type except for P99L (Br − ≥ Cl − = I − ). Although the Pro 99 , Pro 324 , and Pro 1021 mutants produced mature protein, the amount of mature protein was much reduced with the Pro 205 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-channel conductance was decreased in the rank order: wild-type CFTR ≥ P99G > P99L ≥ P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro 205 is critical for correct protein processing, and Pro 99 may contribute either directly or indirectly to the Cl − channel pore.