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Volume 271, Number 25, Issue of June 21, 1996 pp. 14995-15001
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, January 17, 1996, and in revised form, March 21, 1996)

From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

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.

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