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Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.

A point mutation in the simian virus 40 large-T gene, which was generated by mixed oligonucleotide mutagenesis and resulted in the conversion of Lys 128 to Thr, produced a large-T antigen that was detected in the cytoplasm but not the nucleus of cells. Deletions within the surrounding sequence Lys-128Lys-Lys-Arg-Lys-Val-Glu also produce cytoplasmic large-T and define a region of the protein involved in nuclear location.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel located in the apical membrane of epithelia. Although cystic fibrosis (CF) is caused by mutations in a single gene encoding CFTR, the disease has a variable clinical phenotype. The most common mutation associated with cystic fibrosis, deletion of a phenylalanine at position 508 (frequency, 67%), is associated with severe disease. But some missense mutations, for example ones in which arginine is replaced by histidine at residue at 117 (R117H; 0.8%), tryptophan at 334 (0.4%), or proline at 347 (0.5%), are associated with milder disease. These missense mutations affect basic residues located at the external end of the second (M2) and in the sixth (M6) putative membrane-spanning sequences. Here we report that, when expressed in heterologous epithelial cells, all three mutants were correctly processed and generated cyclic AMP-regulated apical Cl- currents. Although the macroscopic current properties were normal, the amount of current was reduced. Patch-clamp analysis revealed that all three mutants had reduced single-channel conductances. In addition, R117H showed altered sensitivity to external pH and had altered single-channel kinetics. These results explain the quantitative decrease in macroscopic Cl- current, and suggest that R117, R334 and R347 contribute to the pore of the CFTR Cl- channel. Our results also suggest why R117H, R334W and R347P produce less severe clinical disease and have implications for our understanding of cystic fibrosis.