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 proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro99, Pro205, Pro324, and Pro1021. 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 = Pro324 > Pro1021 > Pro99 >/= Pro205 mutants. The anion selectivity sequence of the mutants (Br- >/= Cl- > I-) resembled wild-type except for P99L (Br- >/= Cl- = I-). Although the Pro99, Pro324, and Pro1021 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 Pro99 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, Pro205 is critical for correct protein processing, and Pro99 may contribute either directly or indirectly to the Cl- channel pore.
Sheppard
D N
DN
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Travis
S M
SM
Ishihara
H
H
Welsh
M J
MJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000502:physiology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D003550:Cystic Fibrosis / Q000235:genetics / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology*; D004594:Electrophysiology; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008961:Models, Structural; D017354:Point Mutation; D011392:Proline; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
C505032:CFTR protein, human; D018118:Chloride Channels; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D011392:Proline; D000242:Cyclic AMP