metallothionein and Cystic-Fibrosis

The most common mutation in the gene associated with cystic fibrosis (CF) causes deletion of phenylalanine at residue 508 (delta F508) of the gene product called CFTR. This mutation results in the synthesis of a variant CFTR protein that is defective in its ability to traffic to the plasma membrane. Because earlier studies showed delta F508-CFTR retains significant phosphorylation-regulated chloride (Cl-) channel activity, processes capable of restoring the mislocalized delta F508-CFTR to the correct cellular destination may have therapeutic benefit. Here we report one such process that involves overexpression of the mutant protein and appears to result in the escape of a small amount of delta F508-CFTR to the plasma membrane. In recombinant cells where expression of delta F508-CFTR is controlled by the metallothionein promoter, this effect can be brought about by treatment with sodium butyrate. Although cAMP-activated Cl- channel activity could also be detected in immortalized human airway epithelial cells homozygous for the delta F508 mutation at the single cell level, treatment with butyrate did not generate a measurable cAMP-stimulated Cl- current in polarized monolayers of primary CF airway epithelia. However, the observation that overexpression can effect the presence of recombinant delta F508-CFTR at the plasma membrane suggests that perhaps other butyrate-like compounds that are more potent and more specific for the promoter of the CF gene may be efficacious in alleviating the Cl- channel defect associated with CF.

Topics: Animals; Butyrates; Butyric Acid; Cell Line; Cell Membrane; Chloride Channels; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorescent Dyes; Humans; Metallothionein; Mice; Pancreas; Promoter Regions, Genetic; Quinolinium Compounds; Recombinant Proteins; Respiratory System; Tumor Cells, Cultured

A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl(-)-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (delta F508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR.

Topics: Animals; Base Sequence; Cell Line; Chloride Channels; Chlorides; Cloning, Molecular; Codon; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exons; Fibroblasts; Gene Expression; Genetic Vectors; Humans; Ion Channels; Kinetics; Membrane Proteins; Metallothionein; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes; Plasmids; Promoter Regions, Genetic; Restriction Mapping; Transfection